U.S. patent application number 17/286334 was filed with the patent office on 2021-12-09 for method for activation/proliferation of t cells.
This patent application is currently assigned to Takeda Pharmaceutical Company Limited. The applicant listed for this patent is Takeda Pharmaceutical Company Limited. Invention is credited to Akira HAYASHI, Yoshiaki KASSAI, Shinobu KUWAE, Satoru MATSUMOTO, Kazuhide NAKAYAMA.
Application Number | 20210381006 17/286334 |
Document ID | / |
Family ID | 1000005840599 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210381006 |
Kind Code |
A1 |
KUWAE; Shinobu ; et
al. |
December 9, 2021 |
METHOD FOR ACTIVATION/PROLIFERATION OF T CELLS
Abstract
The present invention provides a method for
activating/proliferating T cells, including a step of contacting a
cell population containing T cells with a nucleic acid delivery
carrier having at least one kind of T cell activating ligand added
to its surface, and a method for delivering a nucleic acid into T
cells, the methods including a step of contacting a cell population
containing T cells simultaneously with (a) a nucleic acid delivery
carrier having at least one kind of T cell activating ligand added
to its surface and containing a nucleic acid inside, or (b) at
least one kind of T cell activating ligand, and a nucleic acid
delivery carrier containing a nucleic acid inside and free of a T
cell activating ligand added to its surface, a method for producing
a medicament containing T cells and the like.
Inventors: |
KUWAE; Shinobu; (Kanagawa,
JP) ; MATSUMOTO; Satoru; (Kanagawa, JP) ;
HAYASHI; Akira; (Kanagawa, JP) ; KASSAI;
Yoshiaki; (Kanagawa, JP) ; NAKAYAMA; Kazuhide;
(Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Takeda Pharmaceutical Company Limited |
Chuo-ku, Osaka-shi, Osaka |
|
JP |
|
|
Assignee: |
Takeda Pharmaceutical Company
Limited
Chuo-ku, Osaka-shi, Osaka
JP
|
Family ID: |
1000005840599 |
Appl. No.: |
17/286334 |
Filed: |
October 17, 2019 |
PCT Filed: |
October 17, 2019 |
PCT NO: |
PCT/JP2019/040937 |
371 Date: |
April 16, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/127 20130101;
C07K 16/2818 20130101; C12N 15/88 20130101; A61K 35/17 20130101;
C07K 16/2809 20130101 |
International
Class: |
C12N 15/88 20060101
C12N015/88; A61K 35/17 20060101 A61K035/17; A61K 9/127 20060101
A61K009/127; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 18, 2018 |
JP |
2018-197069 |
Jul 3, 2019 |
JP |
2019-124629 |
Claims
1. A method for activating/proliferating T cells, comprising a step
of contacting a cell population containing T cells with a nucleic
acid delivery carrier having at least one kind of T cell activating
ligand added to its surface.
2. The method according to claim 1, wherein the aforementioned T
cell activating ligand includes an antibody to CD3 and/or an
antibody to CD28.
3. The method according to claim 1, wherein the aforementioned
nucleic acid delivery carrier has two or more kinds of T cell
activating ligands added to a surface thereof.
4. The method according to claim 1, wherein the aforementioned
nucleic acid delivery carrier is a lipid nanoparticle or a
liposome.
5. The method according to claim 1, wherein the aforementioned
nucleic acid delivery carrier comprises, in its inside, a nucleic
acid that suppresses expression of a T cell activation inhibitory
factor and/or a nucleic acid encoding a T cell activation promoting
factor.
6. The method according to claim 1, wherein the aforementioned
nucleic acid delivery carrier comprises a nucleic acid encoding CAR
or TCR.
7. The method according to claim 1, wherein the method is performed
ex vivo.
8. A method for delivering a nucleic acid into T cells, comprising
a step of contacting a cell population containing T cells with a
nucleic acid delivery carrier having at least one kind of T cell
activating ligand added to its surface and containing a nucleic
acid inside, wherein the nucleic acid does not comprise a nucleic
acid encoding CAR or TCR.
9. The method according to claim 8, wherein the aforementioned T
cell activating ligand includes an antibody to CD3 and/or an
antibody to CD28.
10. The method according to claim 8, wherein the aforementioned
nucleic acid delivery carrier has two or more kinds of T cell
activating ligands added to a surface thereof.
11. The method according to claim 8, wherein the aforementioned
nucleic acid delivery carrier is a lipid nanoparticle or a
liposome.
12. The method according to claim 8, wherein the aforementioned
nucleic acid comprises a nucleic acid suppressing expression of a T
cell activation inhibitory factor and/or a nucleic acid encoding a
T cell activation promoting factor.
13. The method according to claim 8, wherein the method is
performed ex vivo.
14. A method for delivering a nucleic acid into T cells, comprising
a step of contacting a cell population containing T cells
simultaneously with at least one kind of T cell activating ligand,
and a nucleic acid delivery carrier containing a nucleic acid
inside and free of a T cell activating ligand added to its
surface.
15. The method according to claim 14, wherein the aforementioned T
cell activating ligand includes an antibody to CD3 and/or an
antibody to CD28.
16. The method according to claim 14, wherein two or more kinds of
T cell activating ligands are contacted.
17. The method according to claim 14, wherein the 5 aforementioned
nucleic acid delivery carrier is a lipid nanoparticle or a
liposome.
18. The method according to claim 14, wherein the aforementioned
nucleic acid includes a nucleic acid suppressing expression of a T
cell activation inhibitory factor and/or a nucleic acid encoding a
T cell activation promoting factor.
19. The method according to claim 14, wherein the aforementioned
nucleic acid comprises a nucleic acid encoding CAR or TCR.
20. The method according to claim 14, wherein the method is
performed ex vivo.
21. A method for producing a medicament comprising T cells,
comprising a step of contacting a cell population containing T
cells simultaneously with at least one kind of T cell activating
ligand, and a nucleic acid delivery carrier containing a nucleic
acid inside and free of a T cell activating ligand added to its
surface.
22. The method according to claim 21, wherein the aforementioned T
cell activating ligand includes an antibody to CD3 and/or an
antibody to CD28.
23. The method according to claim 21, wherein two or more kinds of
T cell activating ligands are contacted.
24. The method according to claim 21, wherein the aforementioned
nucleic acid delivery carrier is a lipid nanoparticle or a
liposome.
25. The method according to claim 21, wherein the aforementioned
nucleic acid delivery carrier comprises a nucleic acid suppressing
expression of a T cell activation inhibitory factor and/or a
nucleic acid encoding a T cell activation promoting factor.
26. The method according to claim 21, wherein the aforementioned
nucleic acid comprises a nucleic acid encoding CAR or TCR.
27. The method according to claim 21, wherein the method is
performed ex vivo.
28. A T cell into which a nucleic acid has been delivered by the
method according to claim 14.
29. A medicament comprising the T cell according to claim 28.
30. A cell culture comprising a cell population containing T cells,
at least one kind of T cell activating ligand, a nucleic acid
delivery carrier without a T cell activating ligand added to the
surface, and a medium.
31. A composition for delivering a nucleic acid to T cells,
comprising at least one kind of T cell activating ligand, and a
nucleic acid delivery carrier without a T cell activating ligand
added to the surface.
32. A kit for delivering a nucleic acid into T cells, comprising at
least one kind of T cell activating ligand, and a nucleic acid
delivery carrier without a T cell activating ligand added to the
surface.
Description
TECHNICAL FIELD
[0001] The present invention relates to a nucleic acid delivery
carrier with a T cell activating ligand added to the surface, a
method for activating and/or proliferating T cells by using the
nucleic acid delivery carrier, a method for delivering a nucleic
acid into T cells, and the like. The present invention also relates
to a method for activating and/or proliferating T cells and a
method for delivering a nucleic acid into T cells, each
characteristically including bringing a T cell activating ligand
and a nucleic acid delivery carrier into simultaneous contact with
T cells.
(BACKGROUND OF THE INVENTION)
[0002] The research and development of cancer immunotherapy using
CAR-T cells or TCR-T cells introduced with a gene of chimeric
antigen receptor (CAR) or T-cell receptor (TCR) derived from cancer
antigen-specific killer T cell is progressing rapidly. Current
CAR-T cell therapy, such as Kymriah (trade name) and Yescarta
(trade name), which were approved in the U.S., generally includes
producing CAR-T cells by introducing CAR genes into T cells
collected from patients ex vivo using virus vectors such as
lentivirus vector, and administering the CAR-T cells to the
patients. However, this method has the problem that the production
cost becomes high due to the cost of cell culture and preparation
of virus vectors because multiple steps are necessary over a long
period of time such as activation/proliferation of T cells,
preparation of virus vectors, gene transfer into T cells, and the
like.
[0003] As a method for introducing CAR into T cells without using a
virus vector, ex vivo or in vivo transfection of CAR into T cells
has been reported which uses nanoparticles containing aggregates of
CAR-encoding plasmid DNA and a cationic polymer that are coated
with a non-cationic polymer conjugated with anti-CD3 antibody
fragments (patent document 1, non-patent document 1), or
nanocarrier containing mesoporous silica encapsulating CAR-encoding
DNA in the pores and coated with a lipid having a surface modified
with an anti-CD3 antibody (patent document 2).
[0004] Apart therefrom, techniques have been reported for
delivering siRNA to a target cell by encapsulating the siRNA of
interest in "lipid nanoparticles (LNP)", which do not have an
internal pore structure and are composed of a cationic lipid, a
non-cationic helper lipid, and a ligand for delivery to the target
cell. For example, ex vivo or in vivo transfection of siRNA for
CD45 into T cells by using an anti-CD4 antibody fragment as a
targeted ligand has been reported (patent document 3, non-patent
document 2).
[0005] In addition, patent document 4 describes a cationic lipid
for introducing an active ingredient such as a nucleic acid or the
like into various cells including T cell, tissues and organs.
[0006] On the other hand, as a method for activating/proliferating
T cells, a method for activating and/or proliferating T cells using
beads on which anti-CD3/CD28 antibody is immobilized or nano-sized
matrix beads has been reported (patent documents 5 and 6).
[0007] However, no technique has been reported heretofore in which
the step of activating/proliferating T cells and the step of
introducing a gene into T cells can be performed simultaneously in
one pod.
DOCUMENT LIST
Patent Documents
[0008] patent document 1: US 2017/0296676 [0009] patent document 2:
US 2016/0145348 [0010] patent document 3: WO 2016/189532 [0011]
patent document 4: WO 2016/021683 [0012] patent document 5: U.S.
Pat. No. 6,352,694 [0013] patent document 6: US 2014/0087462
Non-Patent Documents
[0013] [0014] non-patent document 1: Nature Nanotechnology 12,
813-820 (2017) [0015] non-patent document 2: ACS Nano, 2015, 9(7),
6706-6716
SUMMARY OF INVENTION
Technical Problem
[0016] An object of the present invention is to shorten and
simplify the production process of an agent for immune cell therapy
such as CAR-T therapy and the like, to provide an agent for immune
cell therapy in a short period of time with a low production cost,
and to provide a safer production process of an agent for immune
cell therapy that eliminates a potential risk of carcinogenicity
due to a virus vector.
Solution to Problem
[0017] The present inventors have conducted intensive studies in an
attempt to achieve the above-mentioned object and succeeded in
simultaneously performing a step of activating/proliferating T
cells and a step of introducing a gene into T cells in one pod by
using a nucleic acid delivery carrier having a T cell activating
ligand added to its surface. Furthermore, the present inventors
have surprisingly found that activation/proliferation of T cells
and introduction of nucleic acid into T cells can be efficiently
achieved by simply bringing the T cell activating ligand and the
nucleic acid delivery carrier into contact with the T cells at the
same time, and completed the present invention.
[0018] Accordingly, the present invention provides the following.
[0019] [1] A method for activating/proliferating T cells,
comprising a stepof contacting a cell population containing T cells
with a nucleic acid delivery carrier having at least one kind of T
cell activating ligand added to its surface. [0020] [2] The method
of [1], wherein the aforementioned T cell activating ligand
includes an antibody to CD3 and/or an antibody to CD28. [0021] [3]
The method of [1] or [2], wherein the aforementioned nucleic acid
delivery carrier has two or more kinds of T cell activating ligands
added to a surface thereof. [0022] [4] The method of any of [1] to
[3], wherein the aforementioned nucleic acid delivery carrier is a
lipid nanoparticle or a liposome. [0023] [5] The method of any of
[1] to [4], wherein the aforementioned nucleic acid delivery
carrier comprises, in its inside, a nucleic acid that suppresses
expression of a T cell activation inhibitory factor and/or a
nucleic acid encoding a T cell activation promoting factor. [0024]
[6] The method of any of [1] to [5], wherein the aforementioned
nucleic acid delivery carrier comprises a nucleic acid encoding CAR
or TCR. [0025] [7] The method of any of [1] to [6], wherein the
method is performed ex vivo. [0026] [8] A method for delivering a
nucleic acid into T cells, comprising a step of contacting a cell
population containing T cells with a nucleic acid delivery carrier
having at least one kind of T cell activating ligand added to its
surface and containing a nucleic acid inside. [0027] [9] The method
of [8], wherein the aforementioned T cell activating ligand
includes an antibody to CD3 and/or an antibody to CD28. [0028] [10]
The method of [8] or [9], wherein the aforementioned nucleic acid
delivery carrier has two or more kinds of T cell activating ligands
added to a surface thereof.
[0029] [11] The method of any of [8] to [10], wherein the
aforementioned nucleic acid delivery carrier is a lipid
nanoparticle or a liposome. [0030] [12] The method of any of [8] to
[11], wherein the aforementioned nucleic acid comprises a nucleic
acid suppressing expression of a T cell activation inhibitory
factor and/or a nucleic acid encoding a T cell activation promoting
factor. [0031] [13] The method of any of [8] to [12], wherein the
aforementioned nucleic acid comprises a nucleic acid encoding CAR
or TCR. [0032] [14] The method of any of [8] to [13], wherein the
method is performed ex vivo. [0033] [15] A method for delivering a
nucleic acid into T cells, comprising a step of contacting a cell
population containing T cells simultaneously with at least one kind
of T cell activating ligand, and a nucleic acid delivery carrier
containing a nucleic acid inside and free of a T cell activating
ligand added to its surface. [0034] [16] The method of [15],
wherein the aforementioned T cell activating ligand includes an
antibody to CD3 and/or an antibody to CD28. [0035] [17] The method
of [15] or [16], wherein two or more kinds of T cell activating
ligands are contacted. [0036] [18] The method of any of [15] to
[17], wherein the aforementioned nucleic acid delivery carrier is a
lipid nanoparticle or a liposome. [0037] [19] The method of any of
[15] to [18], wherein the aforementioned nucleic acid includes a
nucleic acid suppressing expression of a T cell activation
inhibitory factor and/or a nucleic acid encoding a T cell
activation promoting factor. [0038] [20] The method of any of [15]
to [19], wherein the aforementioned nucleic acid comprises a
nucleic acid encoding CAR or TCR. [0039] [21] The method of any of
[15] to [20], wherein the method is performed ex vivo. [0040] [22]
A method for producing a medicament comprising T cells, comprising
a step of contacting a cell population containing T cells with a
nucleic acid delivery carrier having at least one kind of T cell
activating ligand added to its surface and containing a nucleic
acid inside. [0041] [23] The method of [22], wherein the
aforementioned T cell activating ligand includes an antibody to CD3
and/or an antibody to CD28. [0042] [24] The method of [22] or [23],
wherein the aforementioned nucleic acid delivery carrier has two or
more kinds of T cell activating ligands added to a surface thereof.
[0043] [25] The method of any of [22] to [24], wherein the
aforementioned nucleic acid delivery carrier is a lipid
nanoparticle or a liposome. [0044] [26] The method of any of [22]
to [25], wherein the aforementioned nucleic acid delivery carrier
comprises a nucleic acid suppressing expression of a T cell
activation inhibitory factor and/or a nucleic acid encoding a T
cell activation promoting factor. [0045] [27] The method of any of
[22] to [26], wherein the aforementioned nucleic acid comprises a
nucleic acid encoding CAR or TCR. [0046] [28] The method of any of
[22] to [27], wherein the method is performed ex vivo. [0047] [29]
A method for producing a medicament comprising T cells, comprising
a step of contacting a cell population containing T cells
simultaneously with at least one kind of T cell activating ligand,
and a nucleic acid delivery carrier containing a nucleic acid
inside and free of a T cell activating ligand added to its surface.
[0048] [30] The method of [29], wherein the aforementioned T cell
activating ligand includes an antibody to CD3 and/or an antibody to
CD28. [0049] [31] The method of [29] or [30], wherein two or more
kinds of T cell activating ligands are contacted. [0050] [32] The
method of any of [29] to [31], wherein the aforementioned nucleic
acid delivery carrier is a lipid nanoparticle or a liposome. [0051]
[33] The method of any of [29] to [32], wherein the aforementioned
nucleic acid delivery carrier comprises a nucleic acid suppressing
expression of a T cell activation inhibitory factor and/or a
nucleic acid encoding a T cell activation promoting factor. [0052]
[35] The method of any of [29] to [34], wherein the method is
performed ex vivo. [0053] [36] A nucleic acid delivery carrier
having at least one kind of T cell activating ligand added to its
surface. [0054] [37] The nucleic acid delivery carrier of [36],
wherein the aforementioned T cell activating ligand includes an
antibody to CD3 and/or an antibody to CD28. [0055] [38] The nucleic
acid delivery carrier of [36] or [37], wherein the aforementioned
nucleic acid delivery carrier has two or more kinds of T cell
activating ligands added to a surface thereof. [0056] [39] The
nucleic acid delivery carrier of any of [36] to [38], wherein the
aforementioned nucleic acid delivery carrier is a lipid
nanoparticle or a liposome. [0057] [40] The nucleic acid delivery
carrier of any of [36] to [39], comprising, in the inside, a
nucleic acid suppressing expression of a T cell activation
inhibitory factor and/or a nucleic acid encoding a T cell
activation promoting factor. [0058] [40] The nucleic acid delivery
carrier of any of [36] to [40], comprising, in the inside, a
nucleic acid encoding CAR or TCR. [0059] [11] A medicament
comprising the nucleic acid delivery carrier of any of [36] to
[41]. [0060] [43] A T cell into which a nucleic acid has been
delivered by the method of any of [15] to [21]. [0061] [44] A
medicament comprising the T cell of [43]. [0062] [45] A cell
culture comprising a cell population containing T cells, at least
one kind of T cell activating ligand, a nucleic acid delivery
carrier without a T cell activating ligand added to the surface,
and a medium. [0063] [46] A composition for delivering a nucleic
acid to T cells, comprising at least one kind of T cell activating
ligand, and a nucleic acid delivery carrier without a T cell
activating ligand added to the surface. [0064] [47] A kit for
delivering a nucleic acid into T cells, comprising at least one
kind of T cell activating ligand, and a nucleic acid delivery
carrier without a T cell activating ligand added to the surface.
[0065] [48] A T cell into which a nucleic acid has been delivered
by the method of any of [8] to [14]. [0066] [49] A medicament
comprising the T cell of [48]. [0067] [50] A cell culture
comprising a cell population containing T cells, a nucleic acid
delivery carrier having at least one kind of T cell activating
ligand added to its surface and containing a nucleic acid inside,
and a medium. [0068] [51] A composition for delivering a nucleic
acid into T cells, comprising a nucleic acid delivery carrier
having at least one kind of T cell activating ligand added to its
surface and containing a nucleic acid inside. [0069] [52] A kit for
delivering a nucleic acid into T cells, comprising a nucleic acid
delivery carrier having at least one kind of T cell activating
ligand added to its surface and containing a nucleic acid
inside.
Advantageous Effects of Invention
[0070] According to the present invention, a step of
activating/proliferating T cells and a step of introducing a gene
into T cells can be performed simultaneously in one pod without
using a virus vector. As a result, an agent for immune cell therapy
can be provided in a short period of time with a low production
cost.
[BRIEF DESCRIPTION OF DRAWINGS]
[0071] FIG. 1 shows a comparison of efficiency of gene transfer
into T cells by lipid nanoparticles having an anti-CD3 antibody
added to surface thereof, and containing various cationic lipids
(compounds 7, 11, 12, 21, 31 and 35).
[0072] FIG. 2 shows a comparison of efficiency of gene transfer
into T cells by lipid nanoparticles having an anti-CD3 antibody
and/or an anti-CD28 antibody added to surface thereof.
[0073] FIG. 3 shows that gene transfer (I) into T cells and
activation (II) of T cells are simultaneously achieved by lipid
nanoparticles having an anti-CD3 antibody and an anti-CD28 antibody
added to surface thereof. In (I) and (II), the numerical value in
the upper panel shows a concentration (.mu.g/ml) of encapsulated
mRNA, and the numerical value in the lower panel shows a
concentration (.mu.g/ml) of the antibody. For comparison, (III)
shows efficiency of T cell activation by beads having
conventionally-known anti-CD3 antibody and anti-CD28 antibody added
to surface thereof.
[0074] FIG. 4 shows that luc mRNA is efficiently introduced into
human peripheral blood CD3-positive pan-T cells by co-addition of
an activation stimulant and lipid nanoparticles.
[0075] FIG. 5 shows the survival and proliferation rate of T cells
transfected with luc mRNA by lipid nanoparticles.
[0076] FIG. 6 shows that luc mRNA is efficiently introduced into
human CD4/CD8-positive T cells by co-addition of an activation
stimulant and lipid nanoparticles (left), and that the survival and
proliferation rate of T cells is maintained at a high level
(right).
DETAILED DESCRIPTION OF THE INVENTION
1. Nucleic Acid Delivery Carrier of the Present Invention
[0077] The present invention provides a nucleic acid delivery
carrier having at least one kind of T cell activating ligand added
to its surface (hereinafter to be also referred to as "the nucleic
acid delivery carrier of the present invention").
[0078] As used herein, the "nucleic acid delivery carrier" means a
carrier capable of supporting a nucleic acid and delivering the
nucleic acid into a cell. Being "capable of delivering the nucleic
acid into a cell" means that a nucleic acid being carried can be
delivered at least into the cytoplasm of a cell.
1-1. Nucleic Acid Delivery Carrier
[0079] The nucleic acid delivery carrier to be used in the present
invention is not particularly limited in terms of the structure
thereof, component molecules, and nucleic acid carrying form as
long as it can support a nucleic acid and can deliver the nucleic
acid into a cell, as described above. A representative drug
delivery system (DDS) of nucleic acid is, for example, a complex
using positively-charged cationic liposomes, cationic polymers, and
the like as carriers, and formed based on the electrostatic
interaction between them and nucleic acid. The complex binds to a
negatively-charged cell membrane and is then incorporated into the
cell by adsorptive endocytosis.
[0080] More specifically, examples of the nucleic acid delivery
carrier to be used in the present invention include, but are not
limited to, lipid nanoparticles (LNP), liposomes (e.g., cationic
liposome, PEG-modified liposome, etc.), and cationic polymers
(e.g., polyethyleneimine, polylysine, polyornithine, chitosan,
atelocollagen, protamine etc.), those in which a cationic polymer
is encapsulated in liposomes, and the like. Alternatively, exosome,
which is a component derived from living organisms, can also be
used. Preferred is lipid nanoparticle or liposome, more preferred
is lipid nanoparticle. [0081] 1-1-1. Lipid Nanoparticle (LNP)
[0082] In the present specification, the "lipid nanoparticle (LNP)"
means a particle with an average diameter of less than 1 .mu.m and
free of a large pore structure (e.g., liposome) or a small pore
structure (e.g., mesoporous material) inside the outer shell of a
lipid aggregate containing cationic lipid and non-cationic
lipid.
[0083] The components of the lipid nanoparticle are described
below.
(a) Cationic Lipid
[0084] In the present specification, the "cationic lipid" means a
lipid that has a net positive charge in a low pH environment such
as in physiological pH, endosome and the like. The cationic lipids
used in the lipid nanoparticle used in the present invention are
not particularly limited. For example, cationic lipids and the like
described in WO 2016/021683, WO 2015/011633, WO 2011/153493, WO
2013/126803, WO 2010/054401, WO 2010/042877, WO 2016/104580, WO
2015/005253, WO 2014/007398, WO 2017/117528, WO 2017/075531, WO
2017/00414, WO 2015/199952, US 2015/0239834, WO2019/131839, and the
like can be mentioned.
[0085] Alternatively, the synthetic cationic lipids (e.g., K-E12,
H-A12, Y-E12, G-O12, K-A12, R-A12, cKK-E12, cPK-E12, PK1K-E12,
PK500-E12, cQK-E12, cKK-A12, KK-A12, PK-4K-E12, cWK-E12, PK500-O12,
PK1K-O12, cYK-E12, cDK-E12, cSK-E12, cEK-E12, cMK-E12, cKK-O12,
cIK-E12, cKK-E10, cKK-E14, and cKK-E16, preferably, cKK-E12,
cKK-E14) described in Dong et al. (Proc Natl Acad Sci U S A. 2014
Apr. 15; 111(15):5753), and the synthetic cationic lipids (e.g.,
C14-98, C18-96, C14-113, C14-120, C14-120, C14-110, C16-96 and
C12-200, preferably 014-110, C16-96 and C12-200) described in Love
KT et al. (Proc Natl Acad Sci U S A. 2010 May 25; 107(21):9915) can
be mentioned.
[0086] In one preferred embodiment, a cationic lipid represented by
the following general formula and described in WO 2016/021683 can
be mentioned.
##STR00001##
wherein
[0087] W is the formula --NR.sup.1R.sup.2 or the formula
--N.sup.+R.sup.3R.sup.4R.sup.5(Z.sup.-),
[0088] R.sup.1 and R.sup.2 are each independently a C.sub.1-4 alkyl
group or a hydrogen atom,
[0089] R.sup.3, R.sup.4 and R.sup.5 are each independently a
0.sub.1-.sub.4 alkyl group,
[0090] Z.sup.- is an anion,
[0091] X is an optionally substituted C.sub.1-5 alkylene group,
[0092] Y.sup.A, Y.sup.B and Y.sup.C are each independently an
optionally substituted methine group,
[0093] L.sup.A, L.sup.B and L.sup.C are each independently an
optionally substituted methylene group or a bond, and
[0094] R.sup.A1, R.sup.A2, R.sup.B1, R.sup.B2, R.sup.C1 and
R.sup.C2 are each independently an optionally substituted
C.sub.4-10 alkyl group, or a salt thereof.
[0095] More preferably, cationic lipids represented by the
following structural formulas can be mentioned.
##STR00002## ##STR00003## ##STR00004##
and salts thereof.
[0096] Among the above-mentioned cationic lipids, more preferred
cationic lipids are represented by the following structural
formulas.
##STR00005##
and salts thereof.
[0097] In another preferred embodiment, a cationic lipid
represented by the following structural formula and described in WO
2019/131839 can be mentioned.
[0098] A compound represented by
##STR00006##
wherein
[0099] n is an integer of 2 to 5,
[0100] R is a linear C.sub.1-5 alkyl group, a linear C.sub.7-11
alkenyl group or a linear C.sub.11 alkadienyl group, and
[0101] wavy lines are each independently shows a cis-type or
trans-type bond,
or a salt thereof.
[0102] More preferably, cationic lipids represented by the
following structural formulas can be mentioned.
##STR00007## ##STR00008## ##STR00009##
and salts thereof.
[0103] Among the above-mentioned cationic lipids, more preferred
cationic lipids are represented by the following structural
formulas.
##STR00010##
and salts thereof.
[0104] In another preferred embodiment, a cationic lipid
represented by the following general formula (III) can be
mentioned.
[0105] A compound represented by
##STR00011##
wherein
[0106] n1 is an integer of 2-6,
[0107] n2 is an integer of 0-2,
[0108] n3 is an integer of 0-2,
[0109] L is --C(O)O-- or --NHC(O)O--,
[0110] Ra is a linear C.sub.5-13 alkyl group, a linear C.sub.13 -17
alkenyl group or a linear C.sub.17 alkadienyl group,
[0111] Rb is a linear C.sub.2-9 alkyl group,
[0112] Rc is a hydrogen atom or a linear C.sub.2-9 alkyl group,
[0113] Rd is a hydrogen atom or a linear C.sub.2-9 alkyl group,
[0114] Re is a linear C.sub.2-9 alkyl group, and
[0115] Rf is a linear C.sub.2-9 alkyl group,
or a salt thereof.
[0116] More preferably, cationic lipids represented by the
following structural formulas can be mentioned.
##STR00012## ##STR00013## ##STR00014## ##STR00015##
##STR00016##
and salts thereof.
[0117] Among the above-mentioned cationic lipids, more preferred
are cationic lipids represented by the following structural
formulas.
##STR00017##
and salts thereof.
[0118] The compound (III) can be produced, for example, by the
following production method. In particular, compound (I) with a
desired structure can be synthesized using appropriate starting
materials according to the structure of the desired compound (III)
in the esterification process. The salt of compound (III) can be
obtained by appropriately mixing with an inorganic base, an organic
base, an organic acid, a basic or an acidic amino acid.
Production Method A (L is --C(O)O--)
##STR00018## ##STR00019## ##STR00020##
[0119] Production Method B (L is --NHC(O)O--)
##STR00021##
[0120] Production Method C
##STR00022## ##STR00023##
[0122] In the above formulas, P.sup.1, P.sup.2, P.sup.3, P.sup.4,
P.sup.5 and P.sup.6 are each independently protecting groups,
compound (A) is the formula:
##STR00024##
compound (B) is the formula:
##STR00025##
R.sup.1 is
##STR00026##
[0123] compound (C) is the formula:
##STR00027##
and R.sup.2 is
##STR00028##
[0125] A starting material or a reagent used in each step in the
above-mentioned production method, as well as the obtained
compound, may each form a salt.
[0126] When the compound obtained in each step is a free compound,
this compound can be converted to a salt of interest by a method
known per se in the art. On the contrary, when the compound
obtained in each step is a salt, this salt can be converted to a
free form or another type of salt of interest by a method known per
se in the art.
[0127] The compound obtained in each step may be used in the next
reaction directly in the form of its reaction solution or after
being obtained as a crude product. Alternatively, the compound
obtained in each step can be isolated and/or purified from the
reaction mixture by a separation approach such as concentration,
crystallization, recrystallization, distillation, solvent
extraction, fractionation, or chromatography according to a routine
method.
[0128] If a starting material or a reagent compound for each step
is commercially available, the commercially available product can
be used directly.
[0129] In the reaction of each step, the reaction time can differ
depending on the reagent or the solvent used and is usually 1 min
to 48 hr, preferably 10 min to 8 hr, unless otherwise
specified.
[0130] In the reaction of each step, the reaction temperature can
differ depending on the reagent or the solvent used and is usually
-78.degree. C. to 300.degree. C., preferably -78.degree. C. to
150.degree. C., unless otherwise specified.
[0131] In the reaction of each step, the pressure can differ
depending on the reagent or the solvent used and is usually 1 atm
to 20 atm, preferably 1 atm to 3 atm, unless otherwise
specified.
[0132] In the reaction of each step, for example, a microwave
synthesis apparatus such as a Biotage Initiator may be used. The
reaction temperature can differ depending on the reagent or the
solvent used and is usually room temperature to 300.degree. C.,
preferably room temperature to 250.degree. C., more preferably
50.degree. C. to 250.degree. C., unless otherwise specified. The
reaction time can differ depending on the reagent or the solvent
used and is usually 1 min to 48 hr, preferably 1 min to 8 hr,
unless otherwise specified.
[0133] In the reaction of each step, the reagent is used at 0.5
equivalents to 20 equivalents, preferably 0.8 equivalents to 5
equivalents, with respect to the substrate, unless otherwise
specified. In the case of using the reagent as a catalyst, the
reagent is used at 0.001 equivalents to 1 equivalent, preferably
0.01 equivalents to 0.2 equivalents, with respect to the substrate.
When the reagent also serves as a reaction solvent, the reagent is
used in the amount for the solvent.
[0134] In each step of a reaction, the reaction is carried out
without a solvent or by dissolution or suspension in an appropriate
solvent, unless otherwise specified. Specific examples of the
solvent include the following.
[0135] alcohols: methanol, ethanol, isopropanol, isobutanol,
tert-butyl alcohol, 2-methoxyethanol and the like;
[0136] ethers: diethyl ether, diisopropyl ether, diphenyl ether,
tetrahydrofuran, 1,2-dimethoxyethane and the like;
[0137] aromatic hydrocarbons: chlorobenzene, toluene, xylene and
the like;
[0138] saturated hydrocarbons: cyclohexane, hexane, heptane and the
like;
[0139] amides: N,N-dimethylformamide, N-methylpyrrolidone and the
like;
[0140] halogenated hydrocarbon s: dichloromethane, carbon
tetrachloride and the like;
[0141] nitriles: acetonitrile and the like;
[0142] sulfoxide: dimethyl sulfoxide and the like;
[0143] aromatic organic bases: pyridine and the like;
[0144] acid anhydrides: acetic anhydride and the like;
[0145] organic acids: formic acid, acetic acid, trifluoroacetic
acid and the like;
[0146] inorganic acids: hydrochloric acid, sulfuric acid and the
like;
[0147] esters: ethyl acetate, isopropyl acetate ester and the
like;
[0148] ketones: acetone, methyl ethyl ketone and the like;
water.
[0149] Two or more of these solvents may be used as a mixture at an
appropriate ratio.
[0150] In each reaction step making use of a base, examples of
bases that may be used are those listed below.
[0151] inorganic bases: sodium hydroxide, potassium hydroxide,
magnesium hydroxide and the like;
[0152] basic salts: sodium carbonate, calcium carbonate, sodium
hydrogen carbonate and the like;
[0153] organic bases: triethylamine, diethylamine, pyridine,
4-dimethylaminopyridine, N,N-dimethylaniline,
1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene,
imidazole, piperidine and the like;
[0154] metal alkoxides: sodium ethoxide, potassium tert-butoxide,
sodium tert-butoxide and the like;
alkali metal hydrides: sodium hydride and the like;
[0155] metal amides: sodium amide, lithium diisopropylamide,
lithium hexamethyldisilazide and the like;
[0156] organic lithiums: n-butyllithium, sec-butyllithium and the
like.
[0157] In each reaction step making use of an acid or acid
catalyst, the following acids or acid catalysts are used.
[0158] inorganic acids: hydrochloric acid, sulfuric acid, nitric
acid, hydrobromic acid, phosphoric acid and the like;
[0159] organic acids: acetic acid, trifluoroacetic acid, citric
acid, p-toluenesulfonic acid, 10-camphor sulfonic acid and the
like;
[0160] Lewis acid: boron trifluoride diethyl ether complex, zinc
iodide, anhydrous aluminum chloride, anhydrous zinc chloride,
anhydrous iron chloride and the like.
[0161] Unless stated otherwise, each reaction step may be carried
out according to a method known per se in the art, such as those
described in Jikken Kagaku Koza (Encyclopedia of Experimental
Chemistry in English), 5th Ed., Vol. 13 to Vol. 19 (edited by the
Chemical Society of Japan); Shin Jikken Kagaku Koza (New
Encyclopedia of Experimental Chemistry in English), Vol. 14 to Vol.
15 (edited by the Chemical Society of Japan); Fine Organic
Chemistry, 2nd Ed. Revised (L. F. Tietze, Th. Eicher, Nankodo);
Organic Name Reactions; The Reaction Mechanism and Essence, Revised
(Hideo Togo, Kodansha); Organic Syntheses Collective Volume I-VII
(John Wiley & Sons, Inc.); Modern Organic Synthesis in the
Laboratory: A Collection of Standard Experimental Procedures (Jie
Jack Li, Oxford University Press); Comprehensive Heterocyclic
Chemistry III, Vol. 1 to Vol. 14 (Elsevier Japan KK); Strategic
Applications of Named Reactions in Organic Synthesis (translated by
Kiyoshi Tomioka, Kagaku-Dojin Publishing); Comprehensive Organic
Transformations (VCH Publishers, Inc.), 1989; etc.
[0162] In each step, the protection or deprotection reaction of a
functional group may be carried out according to a method known per
se in the art, for example, a method described in "Protective
Groups in Organic Synthesis, 4th Ed." (Theodora W. Greene, Peter G.
M. Wuts), Wiley-Interscience, 2007; "Protecting Groups, 3rd Ed."
(P. J. Kocienski) Thieme, 2004); etc.
[0163] Examples of a protective group for a hydroxy group or a
phenolic hydroxy group in alcohols or the like include: ether-type
protective groups such as methoxymethyl ether, benzyl ether,
p-methoxybenzyl ether, t-butyldimethylsilyl ether,
t-butyldiphenylsilyl ether, and tetrahydropyranyl ether; carboxylic
acid ester-type protective groups such as acetic acid ester;
sulfonic acid ester-type protective groups such as methanesulfonic
acid ester; and carbonic acid ester-type protective groups such as
t-butyl carbonate.
[0164] Examples of a protective group for a carbonyl group in
aldehydes include: acetal-type protective groups such as
dimethylacetal; and cyclic acetal-type protective groups such as
cyclic 1,3-dioxane.
[0165] Examples of a protective group for a carbonyl group in
ketones include: ketal-type protective groups such as
dimethylketal; cyclic ketal-type protective groups such as cyclic
1,3-dioxane; oxime-type protective groups such as O-methyloxime;
and hydrazone-type protective groups such as
N,N-dimethylhydrazone.
[0166] Examples of a protective group for a carboxyl group include:
ester-type protective groups such as methyl ester; and amide-type
protective groups such as N,N-dimethylamide.
[0167] Examples of a protective group for thiol include: ether-type
protective groups such as benzyl thioether; and ester-type
protective groups such as thioacetic acid ester, thiocarbonate and
thiocarbamate.
[0168] Examples of a protective group for an amino group or
aromatic heterocycle such as imidazole, pyrrole or indole include:
carbamate-type protective groups such as benzyl carbamate;
amide-type protective groups such as acetamide; alkylamine-type
protective groups such as N-triphenylmethylamine; and
sulfonamide-type protective groups such as methanesulfonamide.
[0169] The protective groups can be removed by use of a method
known per se in the art, for example, a method using an acid, a
base, ultraviolet light, hydrazine, phenylhydrazine, sodium
N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium
acetate or trialkylsilyl halide (e.g., trimethylsilyl iodide or
trimethylsilyl bromide), or a reduction method.
[0170] In each step making use of a reduction reaction, examples of
reducing agents that may be used include: metal hydrides such as
lithium aluminum hydride, sodium triacetoxyborohydride, sodium
cyanoborohydride, diisobutyl aluminum hydride (DIBAL-H), sodium
borohydride and tetramethylammonium triacetoxyborohydride; boranes
such as borane-tetrahydrofuran complex; Raney nickel; Raney cobalt;
hydrogen; and formic acid. For example, Raney-nickel or Raney
cobalt can be used in the presence of hydrogen or formic acid. In
the case of reducing a carbon-carbon double bond or triple bond, a
method using a catalyst such as palladium-carbon or Lindlar's
catalyst may be used.
[0171] In each step making use of an oxidation reaction, examples
of oxidizing agents that may be used include: peracids such as
m-chloroperbenzoic acid (MCPBA), hydrogen peroxide and t-butyl
hydroperoxide; perchlorates such as tetrabutylammonium perchlorate;
chlorates such as sodium chlorate; chlorites such as sodium
chlorite; periodates such as sodium periodate; high-valent iodine
reagents such as iodosylbenzene; manganese reagents, such as
manganese dioxide and potassium permanganate; lead reagents such as
lead tetraacetate; chromium reagents, such as pyridinium
chlorochromate (PCC), pyridinium dichromate (PDC) and Jones'
reagent; halogen compounds such as N-bromosuccinimide (NBS);
oxygen; ozone; sulfur trioxide-pyridine complex; osmium tetroxide;
selenium dioxide; and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ).
[0172] In each step making use of a radical cyclization reaction,
examples of radical initiators that may be used include: azo
compounds such as azobisisobutyronitrile (AIBN); water-soluble
radical initiators such as 4-4'-azobis-4-cyanopentanoic acid
(ACPA); triethylboron in the presence of air or oxygen; and benzoyl
peroxide. Examples of radical initiators to be used include
tributylstannane, tristrimethylsilylsilane,
1,1,2,2-tetraphenyldisilane, diphenylsilane and samarium
iodide.
[0173] In each step making use of a Wittig reaction, examples of
Wittig reagents that may be used include alkylidenephosphoranes.
The alkylidenephosphoranes can be prepared by a method known per se
in the art, for example, the reaction between a phosphonium salt
and a strong base.
[0174] In each step making use of a Horner-Emmons reaction,
examples of reagents that may be used include phosphonoacetic acid
esters such as methyl dimethylphosphonoacetate and ethyl
diethylphosphonoacetate, and bases such as alkali metal hydrides
and organic lithiums.
[0175] In each step making use of a Friedel-Crafts reaction,
examples of reagents that may be used include a Lewis acid and an
acid chloride or alkylating agent (e.g. alkyl halides, alcohols and
olefins). Alternatively, an organic or inorganic acid may be used
instead of the Lewis acid, and acid anhydrides such as acetic
anhydride may be used instead of the acid chloride.
[0176] In each step making use of an aromatic nucleophilic
substitution reaction, a nucleophile (e.g., amine or imidazole) and
a base (e.g., basic salts or organic bases) may be used as
reagents.
[0177] In each step making use of a nucleophilic addition reaction
using a carbanion, nucleophilic 1,4-addition reaction (Michael
addition reaction) using a carbanion, or nucleophilic substitution
reaction using a carbanion, examples of bases that may be used for
generating the carbanion include organolithium reagents, metal
alkoxides, inorganic bases and organic bases.
[0178] In each step making use of a Grignard reaction, examples of
Grignard reagents include aryl magnesium halides such as phenyl
magnesium bromide, and alkyl magnesium halides such as 35 methyl
magnesium bromide, isopropyl magnesium bromide. The Grignard
reagent can be prepared by a method known per se in the art, for
example, the reaction between an alkyl halide or aryl halide and
magnesium metal in ether or tetrahydrofuran as a solvent.
[0179] In each step making use of a Knoevenagel condensation
reaction, an active methylene compound flanked by two
electron-attracting groups (e.g., malonic acid, diethyl malonate or
malononitrile) and a base (e.g., organic bases, metal alkoxides or
inorganic bases) may be used as reagents.
[0180] In each step making use of a Vilsmeier-Haack reaction,
phosphoryl chloride and an amide derivative (e.g.
N,N-dimethylformamide) may be used as reagents.
[0181] In each step making use of an azidation reaction of
alcohols, alkyl halides or sulfonic acid esters, examples of
azidating agents that may be used include diphenylphosphorylazide
(DPPA), trimethylsilylazide and sodium azide. In the case of
azidating, for example, alcohols, a method using
diphenylphosphorylazide and 1,8-diazabicyclo[5,4,0]undec-7-ene
(DBU), a method using trimethylsilylazide and Lewis acid, or the
like can be used.
[0182] In each step making use of a reductive amination reaction,
examples of reducing agents that may be used include sodium
triacetoxyborohydride, sodium cyanoborohydride, hydrogen and formic
acid. When the substrate is an amine compound, examples of carbonyl
compounds that may be used include p-formaldehyde as well as
aldehydes such as acetaldehyde and ketones such as cyclohexanone.
When the substrate is a carbonyl compound, examples of amines that
may be used include primary amines such as ammonia and methylamine,
and secondary amines such as dimethylamine.
[0183] In each step making use of a Mitsunobu reaction,
azodicarboxylic acid esters (e.g. diethyl azodicarboxylate (DEAD)
and diisopropyl azodicarboxylate (DIAD)) and triphenylphosphine may
be used as reagents.
[0184] In each step making use of an esterification, amidation or
ureation reaction, examples of reagents that may be used include
acyl halides such as acid chlorides or acid bromides, and activated
carboxylic acids such as acid anhydrides, active io esters or
sulfate esters. Examples of the activating agents for carboxylic
acids include: carbodiimide condensing agents such as
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD);
triazine condensing agents such as
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride-n-hydrate (DMT-MM); carbonic acid ester condensing agents
such as 1,1-carbonyldiimidazole (CDI); diphenylphosphorylazide
(DPPA); benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (BOP
reagent); 2-chloro-1-methyl-pyridinium iodide (Mukaiyama reagent);
thionyl chloride; lower alkyl haloformate such as ethyl
chloroformate;
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU); sulfuric acid; and combinations
thereof. In the case of using a carbodiimide condensing agent, the
addition of an additive such as 1-hydroxybenzotriazole (HOBt),
N-hydroxysuccinimide (HOSu) or dimethylaminopyridine (DMAP) to the
reaction may be beneficial.
[0185] In each step making use of a coupling reaction, examples of
metal catalysts that may be used include palladium compounds such
as palladium(II) acetate, tetrakis(triphenylphosphine)palladium(0),
dichlorobis(triphenylphosphine)palladium(II),
dichlorobis(triethylphosphine)palladium(II),
tris(dibenzylideneacetone)dipalladium(0),
1,1'-bis(diphenylphosphino)ferrocene palladium(II) chloride and
palladium(II) acetate; nickel compounds such as
tetrakis(triphenylphosphine)nickel(0); rhodium compounds such as
tris(triphenylphosphine)rhodium(III) chloride; cobalt compounds;
copper compounds such as copper oxide and copper(I) iodide; and
platinum compounds. Addition of a base to the reaction may also be
beneficial. Examples of such bases include inorganic bases and
basic salts.
[0186] In each step making use of a thiocarbonylation reaction,
diphosphorus pentasulfide is typically used as a thiocarbonylating
agent. A reagent having a 1,3,2,4-dithiadiphosphetane-2,4-disulfide
structure such as
2,4-bis(4-methoxyphenyl-1,3,2,4-dithiadiphosphetane-2,4-disulfide
(Lawesson reagent) may be used instead of diphosphorus
pentasulfide.
[0187] In each step making use of a Wohl-Ziegler reaction, examples
of halogenating agents that may be used include N-iodosuccinimide,
N-bromosuccinimide (NBS), N- chlorosuccinimide (NCS), bromine and
sulfuryl chloride. The reaction can be accelerated by the further
addition of a radical initiator such as heat, light, benzoyl
peroxide or azobisisobutyronitrile.
[0188] In each step making use of a halogenation reaction of a
hydroxy group, examples of halogenating agents that may be used
include a hydrohalic acid or the acid halide of an inorganic acid;
examples include hydrochloric acid, thionyl chloride, and
phosphorus oxychloride for chlorination and 48% hydrobromic acid
for bromination. In addition, a method for obtaining an alkyl
halide from an alcohol by the action of triphenylphosphine and
carbon tetrachloride or carbon tetrabromide, etc., may also be
used. Alternatively, a method for synthesizing an alkyl halide
through a 2-step reaction involving the conversion of an alcohol to
a sulfonic acid ester and subsequent reaction with lithium bromide,
lithium chloride or sodium iodide may also be used.
[0189] In each step making use of an Arbuzov reaction, examples of
reagents that may be used include alkyl halides such as ethyl
bromoacetate, and phosphites such as triethylphosphite and
tri(isopropyl)phosphite.
[0190] In each step making use of a sulfone-esterification
reaction, examples of the sulfonylating agent used include
methanesulfonyl chloride, p-toluenesulfonyl chloride,
methanesulfonic anhydride and p-toluenesulfonic anhydride and
trifluoromethanesulfonic anhydride.
[0191] In each step making use of a hydrolysis reaction, an acid or
a base may be used as a reagent. In the case of carrying out the
acid hydrolysis reaction of a t-butyl ester, reagents such as
formic acid, triethylsilane or the like may be added to reductively
trap the by-product t-butyl cation.
[0192] In each step making use of a dehydration reaction, examples
of dehydrating agents that may be used include sulfuric acid,
diphosphorus pentoxide, phosphorus oxychloride,
N,N'-dicyclohexylcarbodiimide, alumina and polyphosphoric acid.
[0193] A salt of the compound represented by the above-mentioned
each structural formula is preferably a pharmacologically
acceptable salt. Examples thereof include salts with inorganic
bases (e.g., alkali metal salts such as sodium salt, potassium salt
and the like; alkaline earth metal salts such as calcium salt,
magnesium salt and the like; aluminum salt, ammonium salt), salts
with organic bases (e.g., salts with trimethylamine, triethylamine,
pyridine, picoline, ethanolamine, diethanolamine, triethanolamine,
tromethamine[tris(hydroxymethyl)methylamine], tert-butylamine,
cyclohexylamine, benzylamine, dicyclohexylamine, N,N-dibenzyl
ethylenediamine), salts with inorganic acids (e.g., salts with
hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic
acid, nitric acid, sulfuric acid, phosphoric acid), salts with
organic acids (salts with formic acid, acetic acid, trifluoroacetic
acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid,
maleic acid, citric acid, succinic acid, malic acid,
methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic
acid), salts with basic amino acids (salts with arginine, lysine,
ornithine) or salts with acidic amino acids (salts with aspartic
acid, glutamic acid).
[0194] The ratio (mol %) of the cationic lipid to the total lipids
present in the lipid nanoparticle is, for example, about 10% to
about 80%, preferably about 20% to about 70%, more preferably about
40% to about 60%; however, the ratio is not limited to these.
[0195] Only one kind of the above-mentioned cationic lipid may also
be used or two or more kinds thereof may be used in combination.
When multiple cationic lipids are used, the ratio of the whole
cationic lipid is preferably as mentioned above.
(b) Non-Cationic Lipid
[0196] In the present specification, the "non-cationic lipid" means
a lipid other than the cationic lipid, and is a lipid that does not
have a net positive electric charge at a selected pH such as
physiological pH and the like. Examples of the non-cationic lipid
used in the lipid nanoparticle of the present invention include
phospholipid, steroids, PEG lipid and the like.
[0197] To enhance the delivery of nucleic acid into, for example, T
cell, the phospholipid is not particularly limited as long as it
stably maintains nucleic acid and does not inhibit fusion with
cellular membranes (plasma membrane and organelle membrane). For
example, phosphatidyl choline, phosphatidyl ethanolamine,
phosphatidyl serine, phosphatidyl inositol, phosphatidic acid,
palmitoyloleoylphosphatidyl choline, lysophosphatidyl choline,
lysophosphatidyl ethanolamine, dipalmitoylphosphatidyl choline,
dioleoylphosphatidyl choline, distearoylphosphatidyl choline,
dilinolenoylphosphatidyl choline and the like can be mentioned.
[0198] Preferred phospholipids include distearoylphosphatidyl
choline (DSPC), dioleoylphosphatidyl choline (DOPC),
dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidyl
glycerol (DOPG), palmitoyloleoylphosphatidyl glycerol (POPG),
dipalmitoylphosphatidyl glycerol (DPPG), dioleoyl-phosphatidyl
ethanolamine (DOPE), palmitoyloleoylphosphatidyl choline (POPC),
palmitoyloleoyl-phosphatidyl ethanolamine (POPE), and
dioleoylphosphatidyl ethanolamine 4-(N-maleimide
methyl)-cyclohexane-1-carboxylate (DOPE-mal), more preferably DOPC,
DPPC, POPC, and DOPE.
[0199] The ratio (mol %) of the phospholipid to the total lipids
present in the lipid nanoparticle may be, for example, about 0% to
about 90%, preferably about 5% to about 30%, more preferably about
8% to about 15%.
[0200] Only one kind of the above-mentioned phospholipid may be
used or two or more kinds thereof may be used in combination. When
multiple phospholipids are used, the ratio of the whole
phospholipid is preferably as mentioned above.
[0201] As the steroids, cholesterol, 5.alpha.-cholestanol,
5.beta.-coprostanol, cholesteryl-(2'-hydroxy)-ethylether,
cholesteryl-(4'-hydroxy)-butylether, 6-ketocholestanol,
5.alpha.-cholestane, cholestenone, 5.alpha.-cholestanone,
5.beta.-cholestanone, and cholesteryl decanoate can be mentioned,
preferably cholesterol.
[0202] The ratio (mol %) of the steroids to the total lipids
present in the lipid nanoparticle when steroids are present may be,
for example, about 10% to about 60%, preferably about 12% to about
58%, more preferably about 20% to about 55%.
[0203] Only one kind of the above-mentioned steroid may be used or
two or more kinds thereof may be used in combination. When multiple
steroids are used, the ratio of the whole steroid is preferably as
mentioned above.
[0204] In the present specification, the "PEG lipid" means any
complex of polyethylene glycol (PEG) and lipid. PEG lipid is not
particularly limited as long as it has an effect of suppressing
aggregation of the lipid nanoparticle. For example, PEG conjugated
with dialkyloxypropyl (PEG-DAA), PEG conjugated with diacylglycerol
(PEG-DAG) (e.g., SUNBRIGHT GM-020 (NOF CORPORATION)), PEG
conjugated with phospholipids such as phosphatidylethanolamine
(PEG-PE), PEG conjugated with ceramide (PEG-Cer), PEG conjugated
with cholesterol (PEG-cholesterol), or derivatives thereof, or
mixtures thereof, mPEG2000-1,2-Di-O-alkyl-sn3-carbomoylglyceride
(PEG-C-DOMG),
1-[8'-(1,2-dimyristoyl-3-propanoxy)-carboxamide-3',6-dioxaoctanyl]carbamo-
yl-.omega.-methyl-poly(ethylene glycol) (2KPEG-DMG) and the like
can be mentioned. Preferred PEG lipid includes PEG-DGA, PEG-DAA,
PEG-PE, PEG-Cer, and a mixture of these, more preferably, a PEG-DAA
conjugate selected from the group consisting of a PEG-didecyl
oxypropyl conjugate, a PEG-dilauryl oxypropyl conjugate, a
PEG-dimyristyl oxypropyl conjugate, a PEG-dipalmityl oxypropyl
conjugate, a PEG-distearyl oxypropyl conjugate, and mixtures
thereof.
[0205] In addition to the methoxy group, the maleimide group,
N-hydroxysuccinimidyl group and the like for binding the T cell
activating ligand can be used as the free end of PEG. For example,
SUNBRIGHT DSPE-0201MA (NOF) can be used as a PEG lipid having a
functional group for binding a T cell-activating ligand (sometimes
to be referred to as "terminal reactive PEG lipid" in the present
specification).
[0206] The ratio (mol %) of the PEG lipid to the total lipids
present in the lipid nanoparticle of the present invention may be,
for example, about 0% to about 20%, preferably about 0.1% to about
5%, more preferably about 0.7% to about 2%.
[0207] The ratio (mol %) of the terminal reactive PEG lipid in the
above-mentioned total PEG lipids may be, for example, about 10% to
about 100%, preferably about 30% to about 100%, more preferably
about 40% to about 100%.
[0208] Only one kind of the above-mentioned PEG lipid may be used
or two or more kinds thereof may be used in combination. When
multiple PEG lipids are used, the ratio of the whole PEG lipid is
preferably as mentioned above.
1-1-2. Liposome
[0209] As another preferable nucleic acid delivery carrier to be
used in the present invention, a liposome can be mentioned. As the
liposome, those conventionally used in DDS of nucleic acids to
cells can be similarly used. For example, liposomes prepared by
mixing various cationic lipids (e.g., DOTMA, DOTAP, DDAB, DMRIE
etc.) developed as transfection reagents, and membrane-fused
neutral lipids (e.g., DOPE, cholesterol etc.) that promote release
from endosome are widely used. Liposomes in which functional
molecules such as PEG, pH-responsive membrane fusion peptide,
membrane permeation promoting peptide and the like are added to the
surface of the liposome can also be used.
1-2. T Cell Activating Ligand
[0210] In the nucleic acid delivery carrier of the present
invention, a T cell activating ligand is added to the surface of
the above-mentioned nucleic acid delivery carrier.
[0211] The T cell activating ligand to be used in the present
invention is not particularly limited as long as it is a molecule
that interacts with the surface molecules of T cells to promote
activation and/or proliferation of the T cells. For example,
molecules having a function of specifically binding to CD3, which
is coupled with TCR and responsible for signal transduction via
TCR, and surface molecules CD28, ICOS, CD137, OX40, CD27, GITR,
BAFFR, TACI, BMCA, CD40L and the like, which are known as
co-stimulation factors of T cell activation, and transducing
activation/proliferation signals and co-signals in T cells or
antigen-presenting cells can be mentioned. Such molecule may be a
physiological ligand (or receptor) for the above-mentioned T cell
surface molecule, or may be a non-physiological ligand (or
receptor) having an agonist activity.
[0212] As the non-physiological ligand, an agonist antibody can be
preferably mentioned.
[0213] More preferably, the T cell activating ligand to be used in
the present invention includes an antibody against CD3 and/or an
antibody against CD28. The antibody against CD3 and the antibody
against CD28 each specifically bind to CD3 and CD28 expressed on
target T cells to be induced to activate and/or proliferate (for
example, when target T cell is derived from human, the antibody
against CD3 and the antibody against CD28 are desirably anti-human
CD3 antibody and anti-human CD28 antibody, respectively), and may
be a complete antibody or a fragment thereof (e.g., Fab,
F(ab').sub.2, Fab', scFv, Fv, reduced antibody (rIgG), dsFv, sFv,
diabody, triabody, etc.) as long as they have the ability to
stimulate these surface molecules of T cells and transduce signals
in the T cells. The subclass of the antibody is also not
particularly limited, but is preferably an IgG antibody.
[0214] When an agonist antibody such as an antibody against CD3 or
an antibody against CD28 is used as the T cell activating ligand, a
commercially available anti-CD3 antibody, anti-CD28 antibody, or
the like can also be used as long as it is a complete antibody
molecule, or the antibody can also be isolated from the culture
medium of the cells that produce the antibody. On the other hand,
when the ligand is any of the aforementioned antibody fragments, by
treating a complete antibody with a reducing agent (e.g.,
2-mercaptoethanol, dithiothreitol) or peptidase (e.g., papain,
pepsin, ficin), or by isolating a nucleic acid encoding fragments
of anti-CD3 antibody, anti-CD28 antibody and the like in the same
manner as in obtaining a nucleic acid to be encapsulated in a
nucleic acid delivery carrier to be described later, the antibody
fragment can be recombinantly produced using the same.
[0215] As the T cell activating ligand, only one kind may be used,
or two or more kinds may be used in combination. It is preferable
to combine two or more kinds. When two or more kinds of T cell
activating ligands are used in combination, at least one kind is
preferably an antibody against CD3 or an antibody against CD28,
more preferably an antibody against CD3. Particularly preferably,
both an antibody against CD3 and an antibody against CD28 can be
used as T cell activating ligands.
[0216] When at least an antibody against CD3 and an antibody
against CD28 are used in combination as the T cell activating
ligands, the molar ratio of the two added to the surface of the
nucleic acid delivery carrier of the present invention is 1:4-4:1,
preferably 1:2-2:1.
[0217] When two or more kinds of T cell activating ligands are used
in combination, the T cell activating ligands may be separately
added to the surface of the nucleic acid delivery carrier, or they
may be complexed and added to the surface of the nucleic acid
delivery carrier as long as the T cell activation activity of each
is maintained. For example, when the two kinds of the T cell
activating ligands are antibodies (e.g., antibody against CD3 and
antibody against CD28), they can be provided as bispecific
antibodies known per se.
[0218] In the nucleic acid delivery carrier of the present
invention, the T cell activating ligand may bind to the outer shell
in any manner as long as it is present on the surface of the
nucleic acid delivery carrier. For example, when a lipid
nanoparticle containing a terminally reactive PEG lipid as a
non-cationic lipid is used as a nucleic acid delivery carrier, the
ligand can be added to the terminal of PEG. For example, lipid
nanoparticles labeled with a ligand (antibody) can be prepared by
reacting a PEG lipid with a maleimide group introduced into the
terminus (e.g., SUNBRIGHT DSPE-0200MA) with the thiol group of the
above-mentioned reduced antibody. Even when a liposome modified
with PEG is used as a nucleic acid delivery carrier, the T cell
activating ligand can be added to the surface of the liposome
surface in the same manner.
1-3. Nucleic Acid Contained in the Nucleic Acid Delivery Carrier of
the Present Invention
[0219] The nucleic acid delivery carrier of the present invention
in a form free of a nucleic acid can also be used to induce
activation and/or proliferation of T cells. In one preferred
embodiment, activation and/or proliferation of T cells and delivery
of the nucleic acid into T cells can be performed simultaneously in
one step with the encapsulated nucleic acid. Therefore, in one
preferred embodiment, the nucleic acid delivery carrier of the
present invention further contains a nucleic acid to be delivered
into T cells.
[0220] When the nucleic acid delivery carrier of the present
invention contains a nucleic acid inside, the nucleic acid is not
particularly limited as long as the nucleic acid itself or a
transcript or translation product thereof has a function of
changing T cells into a desired state within the T cells.
1-3-1. Nucleic Acid Suppressing Expression of T Cell Activation
Inhibitory Factor
[0221] In one preferred embodiment, the nucleic acid delivery
carrier of the present invention contains inside a nucleic acid
suppressing expression of a T cell activation inhibitory factor.
The T cell activation inhibitory factor to be the target is not
particularly limited as long as it suppresses activation of T
cells. For example, immune checkpoint factors (e.g., CTLA-4, PD-1,
TIM-3, LAG-3, TGIT, BTLA, VISTA(PD-1H) etc.) which are cell surface
molecules that transmit negative signals to activation and/or
proliferation of T cells upon receipt of stimulation from
antigen-presenting cells or tumor cells, CD160, Cb1-b, endogenous
TCR and the like can be mentioned.
[0222] A nucleic acid suppressing expression of a T cell activation
inhibitory factor may act at any level from transcription level of
gene encoding the factor, post-transcriptional regulation level,
protein translation level, post-translational modification level,
and the like. Therefore, examples of the nucleic acid suppressing
expression of a T cell activation inhibitory factor include a
nucleic acid (e.g., antigene) inhibiting transcription of a gene
encoding the factor, a nucleic acid inhibiting processing from
initial transcripts to mRNA, a nucleic acid inhibiting translation
from mRNA to protein (e.g., antisense nucleic acid, miRNA) or
degrading mRNA (e.g., siRNA, ribozyme, miRNA) and the like. While
the substances acting at any of those levels are preferably used, a
substance that complementarily binds to mRNA to inhibit translation
into protein or degrades mRNA is preferable. As the nucleic acid,
[0223] (a) nucleic acid having RNAi activity against mRNA encoding
the factor, or a precursor thereof, [0224] (b) antisense nucleic
acid against mRNA encoding the factor, [0225] (c) ribozyme nucleic
acid against mRNA encoding the factor, and the like can be
mentioned.
[0226] The nucleotide sequence of mRNA (cDNA) encoding each T cell
activation inhibitory factor is known, and sequence information can
be obtained, for example, from public databases (e.g., NCBI, EMBL,
DDBJ etc.).
(a) Nucleic Acid Having RNAi Activity Against mRNA Encoding T Cell
Activation Inhibitory Factor, or Precursor thereof
[0227] As the nucleic acid having RNAi activity against mRNA
encoding a T cell activation inhibitory factor, double-stranded RNA
consisting of an oligo RNA complementary to the target mRNA and a
complementary strand thereof, i.e., siRNA can be mentioned. The
siRNA can be designed based on the cDNA sequence information of the
target gene, for example, according to the rules proposed by
Elbashir et al. (Genes Dev., 15, 188-200 (2001)). The short hairpin
RNA (shRNA), which is a precursor of siRNA, can be designed by
appropriately selecting any linker sequence (for example, about
5-25 bases) capable of forming a loop structure, and linking the
sense strand and antisense strand of siRNA via the linker
sequence.
[0228] The siRNA and/or shRNA sequences can be searched using
search software provided free of charge on various websites.
Examples of such site include, but are not limited to, siDESIGN
Center provided by Dharmacon [0229]
(http://dharmacon.horizondiscovery.com/jp/design-center/?rdr=true&LangTyp-
e=1041&pageid=17179928204), siRNA Target Finder provided by
GenScript [0230]
(https://www.genscript.com/tools/sirna-target-finder) and the
like.
[0231] In the present specification, microRNA (miRNA) that targets
mRNA encoding a T cell activation inhibitory factor is also defined
as being included in the nucleic acid having RNAi activity against
the mRNA. For miRNA, primary-microRNA (pri-miRNA), which is the
primary transcript, is first transcribed from a gene encoding the
miRNA, then processed by Drosha into precursor-microRNA (pre-miRNA)
of about 70 bases in length having a characteristic hairpin
structure, transported from the nucleus to the cytoplasm, and
further processed by mediation of Dicer to become mature miRNA,
which is taken up by RISC and acts on the target mRNA. Therefore,
pre-miRNA or pri-miRNA, preferably pre-miRNA, can also be used as a
precursor of miRNA.
[0232] miRNA can be searched using target prediction software
provided free of charge on various websites. Examples of such site
include, but are not limited to, TargetScan
(http://www.targetscan.org/vert 72/) published by Whitehead
Institute, USA,
DIANA-micro-T-CDS(http://diana.imis.athena-innovation.gr/DianaTools/index-
.php?r=microT_CDS/index) published by Alexander Fleming Biomedical
Sciences Research Centre, Greece, and the like. Alternatively,
miRNA targeting mRNA encoding a T cell activation inhibitory factor
can also be searched using TarBase
(http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=ta-
rbasev8/index) which is a database relating to miRNA that has been
experimentally proven to act on the target mRNA, and published by
the University of Thessaly, Pasteur Institute and the like.
[0233] The nucleotide molecules that constitute siRNA and/or shRNA,
or miRNA and/or pre-miRNA may be native RNA or DNA. In order to
improve stability (chemical and/or against enzyme) and specific
activity (affinity for RNA), various chemical modifications known
per se can be included.
[0234] siRNA can be prepared according a process comprising
synthesizing a sense strand and an antisense strand of target
sequence on mRNA each with the DNA/RNA automatic synthesizer,
denaturing in a suitable annealing-buffer solution at about 90 to
95.degree. C. for about 1 minute, and annealing at about 30 to
70.degree. C. for about 1 to 8 hours. In addition, siRNA can also
be prepared by synthesizing shRNA which is the precursor of siRNA
and cleaving the shRNA with the use of a dicer. miRNA and pre-miRNA
can be synthesized by a DNA/RNA automatic synthesizer based on the
sequence information thereof.
[0235] In the present specification, a nucleic acid designed to be
able to produce siRNA or miRNA against mRNA encoding a T cell
activation inhibitor in vivo is also defined as being included in
the nucleic acid having RNAi activity against the mRNA. Examples of
such nucleic acid include expression vectors constructed to express
the above-mentioned shRNA or siRNA or miRNA or pre-miRNA, and the
like. As the promoter, a polII promoter (e.g., CMV early-immediate
promoter) can be used. It is a general practice to use a polIII
promoter to ensure accurate transcription of short RNA. Examples of
the polIII promoter include mouse and human U6-snRNA promoters,
human H1-RNase P RNA promoter, human valine-tRNA promoter and the
like. In addition, a sequence in which four or more Ts are
continuous is used as the transcription termination signal. An
expression cassette of miRNA and pre-miRNA can also be produced in
the same manner as in shRNA.
(b) Antisense Nucleic Acid Against mRNA Encoding T Cell Activation
Inhibitory Factor
[0236] The antisense nucleic acid against mRNA encoding a T cell
activation inhibitory factor is a nucleic acid comprising a
nucleotide sequence complimentary to the nucleotide sequence of
mRNA or a part thereof, which has a function of inhibiting the
protein synthesis by binding specifically with the target mRNA to
form a stable duplex. The antisense nucleic acid may be DNA or RNA,
or DNA/RNA chimera. When the antisense nucleic acid is DNA, the
RNA:DNA hybrid formed by the target RNA and the antisense DNA is
recognized by endogenous RNase H, thereby undergoing the selective
degradation of the target RNA. The length of the target region of
the antisense nucleic acid is not particularly limited as long as
the hybridization of the antisense nucleic acid eventually inhibits
the translation into protein, and may be the entire sequence of
mRNA encoding the protein or a partial sequence thereof. A short
one may be about 10 bases, and a long one may be the entire
sequence of mRNA or initial transcript. In addition, the antisense
nucleic acid may be a nucleic acid that inhibits the translation
into a protein by hybridizing with target mRNA or initial
transcript, and it may as well as be the nucleic acid capable of
forming a triplex by binding with these genes which are the
double-stranded DNAs and inhibiting the transcription into RNA
(anti-gene).
[0237] The nucleotide molecule constituting the antisense nucleic
acid may also be modified in the same manner as in the cases of the
above-mentioned siRNA and the like in order to improve stability,
specific activity and the like.
[0238] The antisense oligonucleotide can be prepared by determining
a target sequence of mRNA or initial transcript based on the cDNA
sequence or genomic DNA sequence of the target gene, and
synthesizing its complementary sequence with the use of a
commercially available DNA/RNA automatic synthesizer.
[0239] In the present specification, a nucleic acid designed to be
able to generate an antisense RNA for mRNA encoding a T cell
activation inhibitory factor in vivo is also defined to be included
in an antisense nucleic acid for the mRNA. Such nucleic acid can be
exemplified by an expression vector so constructed as to express
the above-mentioned antisense RNA, or the like. As the promoter, a
polII promoter or a polIII promoter can be appropriately selected
and used according to the length of the antisense RNA to be
transcribed.
(c) Ribozyme Nucleic Acid for mRNA Encoding T Cell Activation
Inhibitory Factor
[0240] A ribozyme nucleic acid capable of specifically cleaving the
internal coding region of the mRNA encoding a T cell activation
inhibitory factor can also be used as a nucleic acid suppressing
the expression of the factor. The "ribozyme" is narrowly-defined as
RNA having enzymatic activity for cleaving nucleic acid, but the
present specification also includes DNA as long as there is a
sequence specific enzymatic activity for cleaving nucleic acid.
Ribozyme nucleic acid with the broadest utility includes
self-splicing RNA which can be found in infectious RNA such as
viroid, a virusoid, etc., and hammer-head type or hairpin type are
known. When ribozyme is used in the form of an expression vector
having DNA which encodes the ribozyme, the ribozyme can be hybrid
ribozyme further coupled with the sequence of modified tRNA so as
to promote transport to cytoplasm of a transcript.
(d) Nucleic Acid Suppressing Expression of T Cell Activation
Inhibitory Factor by Genome Editing
[0241] In a preferred embodiment different from the above-mentioned
(a)-(c), the nucleic acid suppressing expression of a T cell
activation inhibitory factor can be a nucleic acid that can
inactivate (knock out) a gene encoding the factor. As such nucleic
acid, a nucleic acid encoding an artificial nuclease composed of a
nucleic acid sequence recognition module (e.g., CRISPR/Cas9,
ZFmotif, TAL effector etc.) capable of specifically recognizing a
partial nucleotide sequence in the gene as a target, and a nuclease
that introduces double-strand break (DSB) into the gene in the
inside of or near the target sequence can be mentioned. After DSB
introduction, the gene can be knocked out by insertion or deletion
mutation due to a non-homologous end joining (NHEJ) repair error.
Alternatively, gene knockout by homologous recombination (HR)
repair can also be performed by combining with a targeting vector
in which a marker gene (e.g., reporter gene such as fluorescent
protein gene and the like, selection marker gene such as drug
resistance gene and the like) is inserted in the gene sequence. In
addition, the endogenous TCR gene can also be knocked in by HR
repair with an exogenous TCR gene.
1-3-2. Nucleic Acid Encoding T Cell Activation Promoting Factor
[0242] In another preferred embodiment, the nucleic acid delivery
carrier of the present invention contains inside a nucleic acid
encoding a T cell activation promoting factor. The T cell
activation promoting factor of interest includes, for example, T
cell surface molecules (e.g., CD28, ICOS, CD137, OX40, CD27, GITR,
BAFFR, TACI, BMCA, CD40L etc.) to which the aforementioned T cell
activating ligand binds to transduce activation and/or
proliferation signals in T cells, and the like.
[0243] The nucleotide sequence of mRNA (cDNA) encoding each T cell
activation promoting factor is known, and sequence information can
be obtained, for example, from public databases (e.g., NCBI, EMBL,
DDBJ etc.).
[0244] The nucleic acid encoding the T cell activation promoting
factor can be encapsulated, in the form of an expression vector
containing mRNA or DNA encoding the factor, in the nucleic acid
delivery carrier of the present invention. The mRNA encoding the T
cell activation promoting factor can be isolated by a method known
per se, using RNA extracted from T cells as a template, and using a
probe or primer prepared based on the sequence information thereof.
The obtained mRNA may be encapsulated as it is in the nucleic acid
delivery carrier of the present invention, or can be converted into
cDNA and amplified by RT-PCR.
[0245] The obtained DNA encoding a T cell activation promoting
factor can be inserted into an expression vector, preferably a
plasmid vector, containing a functional promoter in T cells, either
as is or after adding a suitable linker and/or nuclear
translocation signal and the like. Examples of the functional
promoter in T cells include, but are not limited to, constitutive
SR.alpha. promoter, SV40 promoter, LTR promoter, CMV
(cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter,
MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex
virus thymidine kinase) promoter and the like in mammalian cells.
In addition, gene promoters such as CD3, CD4, and CD8, which direct
specific expression in T cells, can also be used.
[0246] The mRNA encoding a T cell activation promoting factor can
be prepared by transcription into mRNA in an in vitro transcription
system known per se using an expression vector containing DNA
encoding the factor as a template.
1-3-3. Nucleic Acid Encoding Chimeric Antigen Receptor (Car) or
Exogenous T Cell Receptor (TCR)
[0247] As mentioned above, the nucleic acid delivery carrier of the
present invention encapsulating the nucleic acid makes it possible
to perform activation and/or proliferation of T cells, and delivery
of the nucleic acid into T cells simultaneously in one step.
Therefore, by encapsulating a nucleic acid encoding CAR or TCR in
the nucleic acid delivery carrier of the present invention, a step
of activation/proliferation of T cells and a step of gene transfer
into T cells can be performed simultaneously in one pod.
[0248] That is, in one preferred embodiment, the nucleic acid
delivery carrier of the present invention contains inside a nucleic
acid encoding CAR or TCR.
(a) Nucleic Acid Encoding CAR
[0249] CAR is an artificially constructed hybrid protein containing
the antigen-binding domain (e.g., scFv) of an antibody coupled to a
T cell signal transduction domain. CAR is characterized by the
ability to utilize the antigen-binding property of the monoclonal
antibody to redirect the specificity and responsiveness of T cells
to a selected target in a non-MHC-restricted manner.
Non-MHC-restricted antigen recognition confers on CAR-expressing T
cells the ability to recognize antigens independently of antigen
processing, thereby bypassing the major mechanism of tumor escape.
Furthermore, when expressed in T cells, CAR advantageously does not
dimerize with the endogenous TCR .alpha. chain and .beta.
chain.
[0250] CAR to be encapsulated in the nucleic acid delivery carrier
of the present invention includes an antigen-binding domain of an
antibody that can specifically recognize surface antigens (e.g.,
cancer antigen peptide, surface receptor showing promoted
expression in cancer cells, etc.) that the target T cell should
recognize, an extracellular hinge domain, a transmembrane domain,
and an intracellular T cell signal transduction domain.
[0251] Examples of the surface antigens specifically recognized by
antigen-binding domains include, but are not limited to, surface
receptors showing promoted expression in various cancers (e.g.,
acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder
cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast
cancer, anus, anal canal or anorectal cancer, cancer of the eye,
cancer of the interhepatic bile duct, joint cancer, cervical,
gallbladder or pleural cancer, nose, nasal cavity or middle ear
cancer, oral cancer, vulvar cancer, chronic myelogenous cancer,
colorectal cancer, esophageal cancer, cervical cancer,
fibrosarcoma, gastrointestinal carcinoid tumor, head and neck
cancer (e.g., head and neck squamous cell carcinoma),
hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia
(e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia,
chronic lymphocytic leukemia, acute myelogenous leukemia), liquid
tumor, liver cancer, lung cancer (e.g., non-small cell lung
cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma,
diffuse large B cell lymphoma, follicular lymphoma), malignant
mesothelioma, mastocytoma, melanoma, multiple myeloma,
nasopharyngeal cancer, ovarian cancer, pancreatic cancer;
peritoneal, omentum and mesenteric cancer; pharyngeal cancer,
prostate cancer, rectal cancer, kidney cancer, skin cancer, small
intestinal cancer, soft tissue cancer, solid tumor, gastric cancer,
testicular cancer, thyroid cancer, ureteral cancer and the like,
for example, CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16,
CD20, mesothelin, B-cell mutation antigen, CD123, CD3, prostate
specific membrane antigen (PSMA), CD33, MUC-1, CD138, CD22, GD2,
PD-L1, CEA, chondroitin sulfate proteoglycan-4, IL-13 receptor a
chain, IgG K light chain, and cancer antigen peptides (e.g.,
peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4,
etc.).
[0252] The antigen-binding domain used in the present invention is
not particularly limited as long as it is an antibody fragment that
can specifically recognize the target antigen. Considering the ease
of preparation of CAR, a single-chain antibody (scFv) in which a
light chain variable region and a heavy chain variable region are
linked via a linker peptide is desirable. The configuration of the
light chain variable region and heavy chain variable region in
single-chain antibody is not particularly limited as long as they
can reconstitute a functional antigen-binding domain. They can
generally be designed in the order of light chain variable region,
linker peptide, and heavy chain variable region from the N-terminal
side. As the linker peptide, a known linker peptide typically used
for the production of single-chain antibodies can be used. For
example, DNA encoding light chain variable region and DNA encoding
heavy chain variable region can be prepared by cloning light chain
gene and heavy chain gene respectively from antibody-producing
cells and performing PCR using them as templates, or the like, or
by chemically synthesizing them from the sequence information of
existing antibodies. DNA encoding a single-chain antibody can be
obtained by ligating each obtained DNA fragment with a DNA encoding
linker peptide by an appropriate method. The N-terminal side of the
antigen-binding domain is preferably further added with a leader
sequence to present CAR to the surface of T cell.
[0253] As the extracellular hinge domain and transmembrane domain,
T cell surface molecule-derived domains generally used in the
relevant technical field can be used as appropriate. For example,
they include, but are not limited to, domains derived from
CD8.alpha. and CD28.
[0254] Examples of the intracellular signal transduction domain
include, but not limited to, those having a CD3.zeta. chain, those
further having a co-stimulatory motif such as CD28, CD134, CD137,
Lck, DAP10, ICOS, 4-1BB, and the like between the transmembrane
domain and the CD3' chain, those having two or more co-stimulatory
motifs and the like. Any domains normally used in the relevant
technical field can be used in combination.
[0255] Sequence information of nucleic acids encoding extracellular
hinge domain, transmembrane domain, and intracellular signaling
domain is well known in the relevant technical field. Those of
ordinary skill in the art can easily obtain DNA fragments encoding
each domain from T cells based on such information.
[0256] DNA encoding CAR can be obtained by linking DNA fragments
respectively encoding the thus-obtained antigen binding domain,
extracellular hinge domain, transmembrane domain, and intracellular
signal transduction domain, by a conventional method.
[0257] The obtained DNA encoding CAR can be inserted into an
expression vector, preferably a plasmid vector, containing a
functional promoter in T cells, either as is or after adding a
suitable linker and/or nuclear localization signal and the like.
Examples of the functional promoter in T cells include, but are not
limited to, constitutive SR.alpha. promoter, SV40 promoter, LTR
promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus)
promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes
simplex virus thymidine kinase) promoter and the like in mammalian
cells. In addition, gene promoters such as CD3, CD4, and CD8, which
direct specific expression in T cells, can also be used.
[0258] RNA encoding a CAR, preferably mRNA, can be prepared by
transcription into mRNA in an in vitro transcription system known
per se using an expression vector containing DNA encoding the
above-mentioned CAR as a template.
(b) Nucleic Acid Encoding Exogenous TCR
[0259] In the present specification, the "T cell receptor (TCR)"
means a receptor that consists of dimers of the TCR chain
(.alpha.-chain, .beta.-chain) and recognizes an antigen or the
antigen-HLA (human leukocyte type antigen) (MHC; major
histocompatibility complex) complex and transduces a stimulatory
signal to T cells. Each TCR chain consists of a variable region and
a constant region, and the variable region contains three
complementarity determining regions (CDR1, CDR2, CDR3). The TCR
used in the present invention includes not only those in which the
.alpha. and .beta. chains of the TCR constitute a heterodimer but
also those in which they constitute a homodimer. Furthermore, the
TCR includes those with a part or all of the constant regions
deleted, those with recombined amino acid sequence, and those with
soluble TCR, and the like.
[0260] The "exogenous TCR" means being exogenous to T cell, which
is the target cell of the nucleic acid delivery carrier of the
present invention. The amino acid sequence of the exogenous TCR may
be the same as or different from that of the endogenous TCR
expressed by T cell, which is the target cell of the nucleic acid
delivery carrier of the present invention.
[0261] The nucleic acid encoding TCR encapsulate in the nucleic
acid delivery carrier of the invention is a nucleic acid encoding
the .alpha. chain and .beta. chain of TCR that can specifically
recognize surface antigens (e.g., cancer antigen peptide etc.) to
be recognized by the target T cell.
[0262] The nucleic acid can be prepared by a method known per se.
When the amino acid sequence or nucleic acid sequence of the
desired TCR is known, a DNA encoding the full-length or a part of
the TCR of the present invention can be constructed based on the
sequence by, for example, chemically synthesizing a DNA strand or
an RNA strand, or connecting a synthesized, partially overlapping
oligo-DNA short strand by the PCR method or the Gibson assembly
method.
[0263] When the sequence of the desired TCR is not known, for
example, T cell of interest is isolated from a population of cells
containing the T cell expressing a TCR of interest, and a nucleic
acid encoding the TCR can be obtained from the T cell.
Specifically, a cell population (e.g., PBMC) containing T cells is
collected from an living organism (e.g., human), the cell
population is cultured in the presence of epitopes of cell surface
antigens recognized by the TCR of interest while stimulating the
cell population, and T cell that specifically recognizes cells
expressing the cell surface antigen can be selected from the cell
population by a known method and using, as indices, specificity for
cells expressing the cell surface antigen and cell surface antigens
such as CD8 and CD4. The specificity for cells expressing the cell
surface antigen of T cells can be measured, for example, by
dextromer assay, ELISPOT assay, cytotoxic assay, or the like. The
aforementioned cell population containing T cells is preferably
collected from, for example, an organism having a large number of
cells expressing a cell surface antigen recognized by the TCR of
interest (e.g., patient with a disease such as cancer, or T
cell-containing population contacted with an epitope of the antigen
or dendritic cells pulsed with the epitope).
[0264] The nucleic acid of the present invention can be obtained by
extracting DNA from the aforementioned isolated T cell by a
conventional method, amplifying and cloning the TCR gene based on
the nucleic acid sequence of the constant region of the TCR by
using the DNA as a template. It can also be prepared by extracting
RNA from a cell and synthesizing cDNA by a conventional method, and
performing 5'-RACE (rapid amplification of cDNA ends) with the cDNA
as templates using antisense primers complementary to the nucleic
acids respectively encoding the constant regions of the TCR a chain
and .beta. chain. 5'-RACE may be performed by a known method and
can be performed, for example, using a commercially available kit
such as SMART PCR cDNA Synthesis Kit (manufactured by clontech).
The DNA encoding the .alpha. chain and .beta. chain of the obtained
TCR can be inserted into an appropriate expression vector in the
same way as the DNA encoding the above-mentioned CAR. The DNA
encoding .alpha. chain and the DNA encoding .beta. chain may be
inserted into the same vector or separate vectors. When inserted
into the same vector, the expression vector may express both
strands in a polycistronic or monocistronic manner.
[0265] In the former case, an intervening sequence that permits
polyscystronic expression, such as IRES or FMV 2A, is inserted
between the DNA encoding both strands.
[0266] In addition, RNA encoding each strand of the TCR, preferably
mRNA, can be prepared in the same way as the above-mentioned RNA
encoding CAR, for example, by using the expression vector as a
template.
1-4. T Cell-Targeting Ligand
[0267] The nucleic acid delivery carrier of the present invention
is desirably used to activate and/or proliferate T cells preferably
ex vivo. It also includes an embodiment of in vivo administration
to a subject. In this case, a ligand capable of targeting the
nucleic acid delivery carrier to T cells (hereinafter to be also
referred to as "T cell targeting ligand") is further added to the
surface of the nucleic acid delivery carrier of the present
invention, whereby the targeting efficiency to T cells can be
enhanced.
[0268] The T cell-targeting ligand is not particularly limited as
long as it can specifically recognize surface molecules that are
specifically or highly expressed in T cells. Preferably, it
includes one containing an antigen binding domain of an antibody
against CD3, CD4 or CD8, more preferably, an anti-CD3 antibody.
Here, the "antigen-binding domain" is synonymous with the
antigen-binding domain that constitutes the above-mentioned CAR.
However, since CAR needs to be prepared as a nucleic acid encoding
same, restrictions occur and single-chain antibodies are generally
used in many cases. Since the antigen-binding domain as a T cell
targeting ligand is contained in a protein state in the lipid
nanoparticle of the present invention, not only single-chain
antibodies, but also any other antibody fragments, such as complete
antibody molecules, Fab, F(ab').sub.2, Fab', Fv, reduced antibody
(rIgG), dsFv, sFv, diabody, triabody, and the like, can also be
used preferably. These antibody fragments can be prepared by
treating the complete antibody (e.g., IgG) with a reduced agent
(e.g., 2-mercaptoethanol, dithiothreitol) or peptidase (e.g.,
papain, pepsin, ficin), or by using a genetic recombination
operation.
[0269] When the T-cell targeting ligand is a complete antibody
molecule, commercially available anti-CD3, CD4, CD8 antibodies,
etc. can be used, or the ligand can be isolated from the culture of
the cells producing the antibody. On the other hand, when the
ligand is any one of the aforementioned antigen-binding domain
(antibody fragment), the nucleic acid encoding the antigen-binding
domain, such as anti-CD3, CD4, CD8 antibodies, etc., is isolated in
the same way as in the nucleic acid encoding the antigen-binding
domain constituting the said CAR is obtained, and the
antigen-binding domain can be recombinantly produced using the
same.
2. Production of the Nucleic Acid Delivery Carrier of the Present
Invention
[0270] As a representative example of the nucleic acid delivery
carrier of the present invention, a production example of the
nucleic acid delivery carrier of the present invention using lipid
nanoparticles as a carrier (hereinafter to be also referred to as
"the lipid nanoparticle of the present invention") is explained in
the following. Even when other carriers such as liposomes and the
like are used, the nucleic acid delivery carrier of the present
invention can be obtained in the same manner by appropriately
making changes according to the carrier used.
[0271] The lipid nanoparticle of the present invention can be
produced, for example, by forming lipid nanoparticles by the method
described in U.S. Pat. No. 9,404,127, and chemically binding the T
cell-activating ligand. Alternatively, as described in WO
2016/021683, for example, an organic solvent solution dissolving
cationic lipid and non-cationic lipid is prepared, the organic
solvent solution is mixed with water or a buffer solution
dissolving the nucleic acid to be encapsulated in the lipid
nanoparticles to prepare lipid nanoparticles, and T cell activating
ligand (further, T cell-targeting ligand as necessary when the
lipid nanoparticle of the present invention is used in vivo) is
chemically bound, whereby the lipid nanoparticle can be produced.
The mixing ratio (molar ratio) of cationic lipid, phospholipid,
cholesterol, and PEG lipid is, for example, 40 to 60:0 to 20:0 to
50:0 to 5, but the ratio is not limited thereto. When PEG lipid is
blended as a non-cationic lipid and a T cell-activating ligand is
added to the terminal of PEG, the mixing ratio (molar ratio) of the
PEG lipid and the ligand may be, for example, 20:1 to 1:20. The
above-mentioned PEG lipid may contain terminal reactive PEG at a
ratio (mol %) of about 10% to about 100%. The above-mentioned
mixing can be conducted using a pipette, a micro fluid mixing
system (e.g., Asia microfluidic system (Syrris)). The obtained
lipid particles may be subject to purification by gel filtration,
dialysis or sterile filtration.
[0272] The concentration of the total lipid component in the
organic solvent solution is preferably 0.5 to 100 mg/mL.
[0273] As the organic solvent, for example, methanol, ethanol,
1-propanol, 2-propanol, 1- butanol, tert-butanol, acetone,
acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, or a
mixture thereof can be recited. The organic solvent may contain 0
to 20% of water or a buffer solution. As the buffer solution,
acidic buffer solutions (e.g., acetate buffer solution, citrate
buffer solution) or neutral buffer solutions (e.g.,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, (HEPE) buffer
solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, a
phosphate buffer solution, phosphate buffered saline (PBS)) can be
recited.
[0274] In the case where a micro fluid mixing system is used for
mixing, preference is given to mixing 1 part by volume of an
organic solvent solution with 1 to 5 parts by volume of water or a
buffer solution. In addition, in said system, the flow rate of the
mixture (a mixture solution of an organic solvent solution and
water or a buffer solution) is preferably 0.1 to 10 mL/min, and the
temperature preferably is 15 to 45.degree. C.
[0275] When a lipid particle dispersion is produced as described
above, the dispersion containing cationic lipid, non-cationic
lipid, nucleic acid and T cell activating ligand can be produced by
adding a nucleic acid to be encapsulated in lipid nanoparticles to
water or buffer. Addition of the nucleic acid in a manner to render
the concentration thereof in water or a buffer solution 0.05 to 2.0
mg/mL is preferable.
[0276] In addition, the lipid nanoparticle can also be produced by
admixing a lipid particle dispersion with the nucleic acid by a
method known per se.
[0277] In the lipid nanoparticle of the present invention, the
content of the nucleic acid is preferably 1-20 wt %. The content
can be measured using Quant-iT.TM.Ribogreen.RTM. (Invitrogen). In
the lipid nanoparticle of the present invention, the encapsulation
ratio of the nucleic acid can be calculated based on the difference
in fluorescence intensity in the presence or absence of the
addition of a surfactant (e.g., Triton-X100).
[0278] A dispersion medium can be substituted with water or a
buffer solution by dialysis. For the dialysis, ultrafiltration
membrane of molecular weight cutoff 10 to 20K is used to carry out
at 4.degree. C. to room temperature. The dialysis may repeatedly be
carried out. For the dialysis, tangential flow filtration may be
used.
[0279] The ratio (weight ratio) of the nucleic acid and the lipid
in the lipid nanoparticle of the present invention obtained as
mentioned above is about 0.01 to about 0.2.
[0280] The average particle size of the lipid nanoparticle of the
present invention is preferably 10 to 200 nm. The average particle
size of the lipid particles can be calculated using, for example,
Zetasizer Nano ZS (Malvern Instruments) on cumulant analysis of an
autocorrelation function.
3. Activation/Proliferation Method of T Cells, Delivery Method of
Nucleic Acid into T Cells, and Production Method of Medicament
Containing T Cells, Using T Cell Activating Ligand and Nucleic Acid
Delivery Carrier
[0281] The present invention also provides an activation and/or
proliferation method of T cells using the nucleic acid delivery
carrier of the present invention obtained as mentioned above
(hereinafter to be also referred to as "the
activation/proliferation method of the present invention"). The
method includes a step of contacting a cell population containing T
cells with the nucleic acid delivery carrier of the present
invention. As used herein, the T cell may be a T cell collected
from a living organism (to be also referred to as "ex vivo T cell"
in the present specification"), or T cell in a living organism (to
be also referred to as "in vivo T cell" in the present
specification), with preference given to ex vivo T cell.
[0282] In a different aspect, when the nucleic acid delivery
carrier of the present invention contains a nucleic acid in the
inside, the activation/proliferation method of the present
invention can simultaneously deliver the nucleic acid into T cells.
Therefore, the present invention also provides a delivery method of
nucleic acid into T cells, including a step of contacting a cell
population containing T cells with the nucleic acid delivery
carrier of the present invention. In another embodiment, the
present invention provides a delivery method of nucleic acid into T
cells, including a step of contacting a cell population containing
T cells simultaneously with at least one kind of T cell activating
ligand, and any of the above-mentioned nucleic acid delivery
carriers without a T cell activating ligand added to the surface
(hereinafter the above-mentioned two embodiments are also to be
collectively referred to as "the nucleic acid delivery method of
the present invention"). In the present specification, when a free
T cell activating ligand that is not bound to a nucleic acid
delivery carrier is used, the ligand may be used alone, or as a
complex in which the ligand is bound to a carrier (e.g.,
Dynabeads(R) (Thermo Fisher Scientific company)). As such complex,
a commercially available product (e.g., TransAct (Milteny Biotech
company), Dynabeads Human T-Activator CD3/CD28 (ThermoFisher
Scientific company)) may also be used.
[0283] In another different aspect, T cells activated and/or
proliferated using the nucleic acid delivery carrier of the present
invention, or T cells into which a nucleic acid is delivered can be
used as an agent for immune cell therapy. Therefore, the present
invention also provides a production method of a medicament
containing T cells, including a step of contacting a cell
population containing T cells with the nucleic acid delivery
carrier of the present invention. In another embodiment, the
present invention provides a production method of a medicament
containing T cells, including a step of contacting a cell
population containing T cells simultaneously with at least one kind
of T cell activating ligand, and any of the above-mentioned nucleic
acid delivery carriers without a T cell activating ligand added to
the surface.
[0284] A cell population containing T cells to be brought into
contact with the nucleic acid delivery carrier of the present
invention, or a T cell activating ligand and a nucleic acid
delivery carrier without a T cell activating ligand added to the
surface may be an isolated T cell or, for example, a non-uniform
cell population such as progenitor cells of lymphocytes including
lymphocytes and pluripotent cells as long as it is a cell
population containing T cell or a progenitor cell thereof. In the
present invention, the "lymphocyte" means one of the subtypes of
leukocyte in the immune system of vertebrates. Examples of the
lymphocyte include T cell, B cell, and natural killer cell (NK
cell), preferably, isolated and purified T cell. In the present
invention, the "T cell" is one type of leukocyte found in lymphatic
organs, peripheral blood, and the like, and refers to one category
of lymphocyte characterized by differentiation and maturation
mainly in the thymus gland and expression of TCR. Examples of the T
cell that can be used in the present invention include cytotoxic T
cell (CTL), which is a CD8-positive cell, helper T cell, which is a
CD4-positive cell, regulatory T cell, and effector T cell, and
preferably, cytotoxic T cell.
[0285] The aforementioned lymphocyte can be collected from, for
example, peripheral blood, bone marrow, and umbilical cord blood of
a human or non-human mammal. When ex vivo T cell contacted with the
nucleic acid delivery carrier of the present invention is used for
the treatment of diseases such as cancer, the cell population is
preferably collected from the person to be treated or a donor with
the HLA type matching with that of the subject to be treated.
[0286] A cell population containing T cells to be brought into
contact with the nucleic acid delivery carrier of the present
invention, or a T cell activating ligand and a nucleic acid
delivery carrier without a T cell activating ligand added to the
surface may be a cell population of T cells obtained by
differentiation induction from progenitor cells of lymphocytes
containing pluripotent cells. Examples of the lymphocyte progenitor
cell, including pluripotent cell, include embryonic stem cell (ES
cell), induced pluripotent stem cell (iPS cell), embryonic cancer
cell (EC cell), embryonic germ cell (EG cell), hematopoietic stem
cell, pluripotent progenitor cell that has lost self-renewal
potential (multipotent progenitor: MMP), common myelo-lymphoid
progenitor cell (MLP), myeloid progenitor cell (MP), granulocyte
mononuclear progenitor cell (GMP), macrophage-dendritic cell
progenitor cell (MDP), dendritic cell progenitor cell (DCP) and the
like. Undifferentiated cells such as pluripotent cell and the like
can be differentiated into T cell by a method known per se.
[0287] There is no particular limitation on the method of
contacting the nucleic acid delivery carrier of the present
invention, or T cell activating ligand and a nucleic acid delivery
carrier without a T cell activating ligand added to the surface
with ex vivo T cells, and, for example, the nucleic acid delivery
carrier of the present invention, or T cell activating ligand and a
nucleic acid delivery carrier without a T cell activating ligand
added to the surface may be added to a typical medium for T cells.
Therefore, in another aspect, the present invention also provides a
cell culture containing a cell population containing T cells, the
nucleic acid delivery carrier of the present invention, or at least
one kind of T cell activating ligand and a nucleic acid delivery
carrier without a T cell activating ligand added to the surface,
and a medium.
[0288] In the nucleic acid delivery method of the present
invention, to increase the efficiency of the delivery nucleic acid,
for example, the calcium phosphate co-precipitation method, PEG
method, electroporation method, microinjection method, lipofection
method, and the like may be used in combination.
[0289] In the nucleic acid delivery method of the present
invention, when the nucleic acid delivery carrier of the present
invention, or a nucleic acid delivery carrier without a T cell
activating ligand added to the surface particularly contains a
nucleic acid encoding exogenous TCR in the inside, the expression
of endogenous TCR .alpha. chain and TCR .beta. chain that are
inherently expressed by the T cell may be suppressed by siRNA from
the viewpoint of an increase in the expression of exogenous TCR,
inhibition of the appearance of mispaired TCR, or inhibition of
self-reactivity. When the above-mentioned nucleic acid is applied
to the method, to avoid the effect of siRNA on exogenous TCR, the
base sequence of a nucleic acid encoding the TCR is preferably a
sequence (codon conversion type sequence) different from the base
sequence corresponding to RNA on which siRNA, which suppresses the
expression of endogenous TCR.alpha. and TCR.beta. chains, acts. The
method therefor is described, for example, in WO 2008/153029. The
aforementioned base sequence can be produced by introducing a
silent mutation into a naturally acquired nucleic acid encoding TCR
or chemically synthesizing an artificially designed nucleic acid.
Alternatively, to avoid mispair with the endogenous TCR chain, a
part or all of the constant regions of the nucleic acid encoding
the exogenous TCR may be replaced with a constant region derived
from an animal other than human, for example, a mouse.
[0290] Alternatively, as mentioned above, endogenous TCR gene may
also be knocked out using a genome editing technique.
4. Nucleic Acid Delivery System Containing at Least One Kind of T
Cell Activating Ligand, a Nucleic Acid Delivery Carrier without a T
Cell Activating Ligand Added to the Surface in Combination
[0291] As mentioned above, a nucleic acid can be delivered into T
cells by contacting a cell population contacting T cells
simultaneously with at least one kind of T cell activating ligand,
and any of the above-mentioned nucleic acid delivery carriers
without a T cell activating ligand added to the surface. Therefore,
the present invention also provides a nucleic acid delivery system
containing at least one kind of T cell activating ligand, and a
nucleic acid delivery carrier without a T cell activating ligand
added to the surface in combination. In the nucleic acid delivery
system, the T cell activating ligand and the nucleic acid delivery
carrier without a T cell activating ligand added to the surface may
be provided as a composition containing the both, or in the form of
a kit containing the both as separate components. The nucleic acid
delivery kit may further contain, in addition to the
above-mentioned both components, for example, a medium to be used
in contacting a cell population containing T cells with such
components, and the like, though not limited to these.
5. Medicament Containing Ex Vivo T Cells Activated and/or
Proliferated by T Cell Activating Ligand and Nucleic Acid Delivery
Carrier, or Delivered with Nucleic Acid
[0292] The present invention also provides ex vivo T cells
activated and/or proliferated by the activation/proliferation
method of the present invention, ex vivo T cells having a nucleic
acid delivered by the nucleic acid delivery method of the present
invention (including cell culture containing medium), and a
medicament containing them.
[0293] The ex vivo T cells activated and/or proliferated by the
activation/proliferation method of the present invention
specifically recognize cells expressing surface antigen
specifically recognized by TCR expressed in the T cells and kill
them (e.g., induction of apoptosis). In addition, the ex vivo T
cells into which a nucleic acid encoding CAR or exogenous TCR is
delivered by the nucleic acid delivery method of the present
invention express the CAR or exogenous TCR, specifically recognize
cells expressing surface antigen specifically recognized by the CAR
or exogenous TCR and can kill them (e.g., induction of apoptosis).
Therefore, ex vivo T cells in which T cells that express TCR
recognizes a surface molecule that is specifically expressed as a
surface antigen or showing enhanced expression in a disease cells,
such as a cancer cell and the like, are activated and/or
proliferated, and ex vivo T cells introduced with a nucleic acid
encoding CAR or exogenous TCR that recognizes the surface molecule
can be used for the prophylaxis or treatment of diseases such as
cancer and the like, and can be safely administered to mammals
(human or other mammal (e.g., mouse, rat, hamster, rabbit, cat,
dog, bovine, sheep, monkey, preferably human)).
[0294] In a medicament containing, as an active ingredient, ex vivo
T cells contacted with the nucleic acid delivery carrier of the
present invention, or T cell activating ligand and a nucleic acid
delivery carrier without a T cell activating ligand added to the
surface, the T cells may be cultured using an appropriate medium
before administration to a subject. In addition, the activation
and/or proliferation of T cells can also be maintained or extended
by adding a stimulation molecule to the medium. Furthermore, serum
or plasma may be added to the medium. While the amount of addition
to these media is not particularly limited, 0% by volume -20% by
volume can be mentioned. Moreover, the amount of serum or plasma to
be used can be changed according to the culturing stage. For
example, serum or plasma concentration can be reduced stepwise. The
origin of serum or plasma may be either autologous or
non-autologous, and autologous one is preferable from the aspect of
safety.
[0295] The medicament containing, as an active ingredient, ex vivo
T cells contacted with the nucleic acid delivery carrier of the
present invention, or T cell activating ligand and a nucleic acid
delivery carrier without a T cell activating ligand added to the
surface is preferably used by parenteral administration to the
subject. Examples of the method for parenteral administration
include intravenous, intraarterial, intramuscular, intraperitoneal,
and subcutaneous administration and the like. While the dose is
appropriately selected according to the condition, body weight, age
and the like of the subject, the medicament is generally
administered such that the cell number is generally
1.times.10.sup.6-1.times.10.sup.10 cells, preferably
1.times.10.sup.7-1.times.10.sup.9 cells, more preferably
5.times.10.sup.7-5.times.10.sup.8 cells, per dose to a subject with
body weight 60 kg. The medicament may be administered once, or in
multiple divided portions. The medicament containing, as an active
ingredient, ex vivo T cells contacted with the nucleic acid
delivery carrier of the present invention can be formulated into a
known form suitable for parenteral administration, for example,
agent for injection or infusion. The medicament of the present
invention may contain pharmacologically acceptable excipients as
appropriate. The pharmacologically acceptable excipient includes
those described above. The medicament may contain saline, phosphate
buffered saline (PBS), medium and the like to maintain the cells
stably. Examples of the medium include, but are not limited to,
media such as RPMI, AIM-V, X-VIVO10 and the like. The medicament
may be supplemented with a pharmaceutically acceptable carrier
(e.g., human serum albumin), preservative and the like for
stabilizing purposes.
[0296] The medicament containing, as an active ingredient, ex vivo
T cells contacted with the nucleic acid delivery carrier of the
present invention, or T cell activating ligand and a nucleic acid
delivery carrier without a T cell activating ligand added to the
surface can be a prophylactic or therapeutic drug for cancer. The
cancer to be the application target for the medicament of the
present invention is not particularly limited. Examples thereof
include, but are not limited to, acute lymphocytic cancer, alveolar
rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g.,
medulloblastoma), breast cancer, anus, anal canal or anorectal
cancer, cancer of the eye, cancer of the interhepatic bile duct,
joint cancer, cervical, gallbladder or pleural cancer, nose, nasal
cavity or middle ear cancer, oral cancer, vulvar cancer, chronic
myelogenous cancer, colon cancer, esophageal cancer, cervical
cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and
neck cancer (e.g., head and neck squamous cell carcinoma),
hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia
(e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia,
chronic lymphocytic leukemia, acute myeloid leukemia), liquid
tumor, liver cancer, lung cancer (e.g., non-small cell lung
cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma,
diffuse large B cell lymphoma, follicular lymphoma), malignant
mesothelioma, mastocytoma, melanoma, multiple myeloma,
nasopharyngeal cancer, ovarian cancer, pancreatic cancer;
peritoneal, omentum and mesenteric cancer; pharyngeal cancer,
prostate cancer, rectal cancer, renal cancer, skin cancer, small
intestine cancer, soft tissue cancer, solid tumor, gastric cancer,
testicular cancer, thyroid cancer, ureteral cancer and the
like.
6. Medicament Containing the Nucleic Acid Delivery Carrier of the
Present Invention
[0297] The nucleic acid delivery carrier of the present invention
can induce activation and/or proliferation of T cells in living
organisms by in vivo administration to mammals such as human and
the like. In addition, by in vivo administration of the nucleic
acid delivery carrier of the present invention containing inside a
nucleic acid encoding CAR or exogenous TCR to mammals such as human
and the like, the nucleic acid is delivered and expressed in T
cells in the living organism, which in turn can impart the T cells
with an ability to specifically recognize cells (e.g., cancer
cells) expressing surface antigen (e.g., cancer antigen)
specifically recognized by CAR or exogenous TCR and kill them
(e.g., induction of apoptosis). Therefore, the present invention
also provides a medicament containing the nucleic acid delivery
carrier of the present invention.
[0298] A medicament containing the nucleic acid delivery carrier of
the present invention is preferably prepared as a pharmaceutical
composition by mixing the nucleic acid delivery carrier with known
pharmaceutically acceptable carriers (including excipient, diluent,
bulking agent, binder, lubricant, flow aid, disintegrant,
surfactant, and the like) and conventional additives, and the like.
The excipients are well known to those of ordinary skill in the art
and include, for example, phosphate-buffered saline (e.g., 0.01M
phosphate, 0.138M NaCl, 0.0027M KCl, pH 7.4), aqueous solutions
containing mineral acid salts such as hydrochloride, hydrobromate,
phosphate, sulfate, and the like, saline solutions, solutions of
glycol, ethanol, and the like, and salts of organic acids such as
acetate, propionate, malonate, benzoate, and the like. In addition,
adjuvants such as wetting agent or emulsifier, and pH buffering
agents can also be used. In addition, preparation adjuvants such as
suspension agent, preservative, stabilizer and dispersing agent may
also be used. Alternatively, the above-mentioned pharmaceutical
composition may be in a dry form which is reconstituted with a
suitable sterile liquid prior to use. The pharmaceutical
composition may be orally or parenterally administered systemically
or topically, depending on the form in which it is prepared (oral
agents such as tablet, pill, capsule, powder, granule, syrup,
emulsion, suspension and the like; parenteral agents such as
injection, drip transfusion, external preparation, suppository and
the like). For parenteral administration, intravenous
administration, intradermal administration, subcutaneous
administration, rectal administration, transdermal administration
and the like are available. When used in an injectable form,
acceptable buffering agent, solubilizing agent, isotonic agent and
the like can also be added.
[0299] The dosage of the medicament of the present invention
containing the nucleic acid delivery carrier of the present
invention is, for example, in the range of 0.001 mg to 10 mg as the
amount of a nucleic acid encoding CAR or exogenous TCR, per 1 kg
body weight per dose. For example, when administered to a human
patient, the dosage is in the range of 0.0001 to 50 mg for a
patient weighing 60 kg. The above-mentioned dosage is an example,
and the dosage can be appropriately selected according to the type
of nucleic acid to be used, administration route, age, weight,
symptoms, etc. of the subject of administration or patient.
[0300] By administration to a mammal (e.g., human or other mammal
(e.g., mouse, rat, hamster, rabbit, cat, dog, bovine, sheep,
monkey), preferably, human), a medicament containing the nucleic
acid delivery carrier of the present invention can induce the
expression of CAR or exogenous TCR in T cell (in vivo T cell) in
the body of the animal. The in vivo T cells kill diseased cells
such as cancer cells and the like expressing surface antigen
targeted by CAR or exogenous TCR, thereby demonstrating a
prophylactic or therapeutic effect against the disease.
[0301] A medicament containing the nucleic acid delivery carrier of
the present invention may be a prophylactic or therapeutic drug for
cancer. The cancer to be the application target of the medicament
of the present invention is not particularly limited. Examples
thereof include, but are not limited to, acute lymphocytic cancer,
alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain
cancer (e.g., medulloblastoma), breast cancer, anus, anal canal or
anorectal cancer, cancer of the eye, cancer of the interhepatic
bile duct, joint cancer, cervical, gallbladder or pleural cancer,
nose, nasal cavity or middle ear cancer, oral cancer, vulvar
cancer, chronic myelogenous cancer, colorectal cancer, esophageal
cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid
tumor, head and neck cancer (e.g., head and neck squamous cell
carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer,
leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic
leukemia, chronic lymphocytic leukemia, acute myelogenous
leukemia), liquid tumor, liver cancer, lung cancer (e.g., non-small
cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin
lymphoma, diffuse large B cell lymphoma, follicular lymphoma),
malignant mesothelioma, mastocytoma, melanoma, multiple myeloma,
nasopharyngeal cancer, ovarian cancer, pancreatic cancer;
peritoneal, omentum and mesenteric cancer; pharyngeal cancer,
prostate cancer, rectal cancer, kidney cancer, skin cancer, small
intestinal cancer, soft tissue cancer, solid tumor, gastric cancer,
testicular tumor, thyroid cancer, ureteral cancer and the like.
[0302] The present invention is explained in more detail in the
following by referring to Examples which are mere exemplifications
and do not limit the present invention.
EXAMPLE
1. Reduction Treatment of Antibody
[0303] 9.21 mg/ml anti-CD3 antibody and anti-CD28 antibody (Bio X
Cell company) solutions (each 111 .mu.l) were mixed with 10 mM DTT
aqueous solution (12.3 .mu.l). The mixture of each antibody and DTT
was mixed by vortex to carry out reaction at room temperature for
30 min. The reaction mixture was fractionated by HPLC (column:
TSKgel G2000SWXL 7.8 mm.times.30 cm, TOSOH, mobile phase: PBS) to
give a fraction solution containing the reduced antibody. The
fraction solution was concentrated by ultrafiltration using Amicon
0.5 ml-10K. The concentrations of the antibody protein and thiol
group in the concentrates were measured by absorbance at 230 nm and
a fluorescence colorimetric reaction with
N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM),
respectively.
2. Preparation of Maleimide-Lipid Nanoparticles
[0304] A lipid mixture containing compound 7, 11, 12, 21, 31 or 35
as cationic lipid (cationic lipid:DPPC:Cholesterol:SUNBRIGHT
GM-020:SUNBRIGHT DSPE-020MA=60:10.6:28:1.4:1, molar ratio) was
dissolved in 90% EtOH, 10% water to give a 7.0 mg/ml lipid
solution. On the other hand, luciferase mRNA (TriLink company) was
dissolved in 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0)
to give 0.2 mg/ml mRNA solution. The obtained lipid solution and
mRNA solution were mixed at room temperature by a Nanoassemblr
apparatus (Precision Nanosystems) at a flow rate ratio of 3
ml/min:6 ml/min to give a dispersion containing the composition.
The obtained dispersion was dialyzed using Slyde-A-Lyzer (20k
fraction molecular weight, Thermo Scientific) against water at room
temperature for 1 hr, and against PBS at 4.degree. C. for 48 hr.
Successively, the dialysate was filtered through a 0.2 .mu.m
syringe filter (Iwaki) and stored at 4.degree. C.
3. Binding Reaction of Reduced Antibody and Maleimide-Lipid
Nanoparticles
[0305] Maleimide-lipid nanoparticle dispersion was mixed with
reduced antibody solution such that the molar concentration of
reduced antibody was 1/20 of that of maleimide, and allowed to
stand at room temperature for 4 hr. Thereafter, the mixture was
stored at 4.degree. C. until the purification step.
4. Gel Filtration Purification of Antibody-Lipid Nanoparticles
[0306] A reaction mixture of a reduced antibody and Maleimide-lipid
nanoparticles was loaded on a gel filtration column Sepharose CL-4B
(Cat No. 17-0150-01/GE Healthcare), and fractionated with D-PBS(-)
as a mobile phase. Successively, the protein concentration of each
fraction was measured to identify the fraction containing the
antibody-lipid nanoparticles of interest, whereby antibody-lipid
nanoparticles stock solution was obtained. The antibody-lipid
nanoparticles were filtered through a 0.2 .mu.m syringe filter and
stored at 4.degree. C.
5. Particle Size Measurement of Antibody-Lipid Nanoparticles
[0307] To 1 .mu.l of an antibody-lipid nanoparticle stock solution
was added 99 .mu.l of phosphate buffered saline (137 mM NaCl, 7.99
mM Na.sub.2HPO.sub.4, 2.7 mM KCl, 1.47 mM KH.sub.2PO.sub.4, pH
7.4). The obtained dispersion was subjected to dynamic light
scattering measurement using Zetasizer Nano ZS (Malvern
instruments), and the cumulant analysis of the autocorrelation
function was performed to measure the Z average particle size and
the polydispersity index (PDI).
6. .dbd. Electric Potential Measurement of Antibody-Lipid
Nanoparticles
[0308] To 50 .mu.l of an antibody-lipid nanoparticle stock solution
was added 700 .mu.l of HEPES buffer (10 mM HEPES-NaOH, pH 7.3). The
obtained dispersion was subjected to electrophoretic light
scattering measurement using Zetasizer Nano ZS (Malvern
instruments) to measure the electric potential.
7. Measurement of Mrna Encapsulation Rate and Concentration of
Antibody-Lipid Nanoparticles
[0309] An antibody-lipid nanoparticle stock solution was diluted
with TE buffer to adjust the mRNA concentration to about 4
.mu.g/ml. As an mRNA concentration standard solution, naked mRNA
was diluted with TE buffer to 4 .mu.g/ml. The diluted
antibody-lipid nanoparticles and naked mRNA concentration standard
solution (each 60 .mu.l) were each mixed with 60 .mu.l of TE buffer
or TE buffer containing 2% Triton-X100. The mixture was allowed to
stand at room temperature for 5 min, mixed with 120 .mu.l of
Quant-iT.TM. RiboGreen (registered trade mark), and the mixture was
further allowed to stand for 5 min. The fluorescence intensity of
the mixture was measured using Envision microplate reader
(Perkin-Elmer company). The mRNA encapsulation rate and mRNA
concentration were calculated by the following formulas.
% mRNA encapsulation rate=(1-F.sub.TE/F.sub.Triton).times.100
mRNA concentration=(F.sub.Triton-b).times.d/m
(wherein F.sub.TE shows RiboGreen fluorescence intensity of lipid
nanoparticles mixed with TE buffer, F.sub.Triton shows RiboGreen
fluorescence intensity of lipid nanoparticles mixed with TE buffer
containing 2% Triton-X100, b and m show y-intercept and slope
obtained from the calibration curve of the concentration standard
siRNA, and d is the dilution rate of lipid nanoparticles)
[0310] A list of the analysis results of the prepared
antibody-lipid nanoparticles is shown in the following Table.
TABLE-US-00001 TABLE 1 mRNA mRNA antibody particle poly- encapsula-
concentra- concentra- size dispersity tion rate tion tion (nm)
index (%) (.mu.g/ml) (.mu.g/ml) anti-CD3- 78 0.114 95 94.1 20.4
compound 7 anti-CD3- 78 0.071 97 87.0 29.4 compound 11 anti-CD3- 99
0.134 96 93.9 27.6 compound 12 anti-CD3- 135 0.121 90 92.3 49.6
compound 31 anti-CD3- 89 0.095 96 73.1 72.7 compound 21 anti-CD3-
108 0.129 97 72.1 65.3 compound 35 anti-CD28- 93 0.137 94 128 10.1
compound 12 anti-CD3/ 97 0.068 98 30.7 55.1 anti-CD28- compound
12
8. mRNA Transfection Into Human Primary Cultured T Cells with Lipid
Nanoparticles Bound to Anti-CD3 Antibody
[0311] Human peripheral blood CD3 positive pan T cells (Precision
Bioservices) were seeded on a round-bottomed 96-well plate
(Corning) at a cell density of 1.times.10.sup.5 cells/well. A
serum-free hematopoietic cell medium X-VIV010 (Lonza) supplemented
with 30 ng/ml recombinant IL-2 (Thermo Fisher Scientific) was used
as the medium. Subsequently, anti-CD3 antibody-bound lipid
nanoparticles encapsulating luciferase mRNA (TriLink) were added to
the medium such that the concentration of luciferase mRNA in the
medium was 1 .mu.g/ml, and the mixture was stood in a 5% CO.sub.2
incubator at 37.degree. C. for 72 hr. Luciferase expressed in T
cells was measured using Bright-Glo Luciferase Assay System Kit
(Promega). The relative luciferase luminescence intensity of T
cells supplemented with each anti-CD3 antibody-lipid nanoparticle
is shown in FIG. 1.
9. mRNA Transfection Into Human Primary Cultured T Cells with Lipid
Nanoparticles Bound to Anti-CD3 Antibody and/or Anti-CD28
Antibody
[0312] Human peripheral blood CD3 positive pan T cells (Precision
Bioservices) were seeded on a round-bottomed 96-well plate
(Corning) at a cell density of 1.times.10.sup.5 cells/well. A
serum-free hematopoietic cell medium X-VIVO10 (Lonza) supplemented
with 30 ng/ml recombinant IL-2 (Thermo Fisher Scientific) was used
as the medium. Subsequently, anti-CD3 antibody-bound lipid
nanoparticles encapsulating luciferase mRNA (TriLink), anti-CD28
antibody-bound lipid nanoparticles, a mixture of anti-CD3
antibody-bound lipid nanoparticles and anti-CD28 antibody-bound
lipid nanoparticles, and lipid nanoparticles bound with a mixture
of anti-CD3 antibody and anti-CD28 antibody were added to the
medium such that the concentration of luciferase mRNA in the medium
was 1 .mu.g/ml, and the mixture was stood in a 5% CO.sub.2
incubator at 37.degree. C. for 72 hr. Luciferase expressed in T
cells was measured using Bright-Glo Luciferase Assay System Kit
(Promega). The relative luciferase luminescence intensity of T
cells supplemented with each antibody-lipid nanoparticle is shown
in FIG. 2.
10. mRNA Transfection Into Human Primary Cultured T Cells with
Lipid Nanoparticles Bound to Anti-CD3/Anti-CD28 Antibody and T Cell
Activation Stimulation
[0313] Human peripheral blood CD3 positive pan T cells (Precision
Bioservices) were seeded on a round-bottomed 96-well plate
(Corning) at a cell density of 1.times.10.sup.5 cells/well. A
serum-free hematopoietic cell medium X-VIVO10 (Lonza) supplemented
with 30 ng/ml recombinant IL-2 (Thermo Fisher Scientific) was used
as the medium. Subsequently, lipid nanoparticles encapsulating
luciferase mRNA (TriLink) and bound to anti-CD3 antibody and
anti-CD28 antibody were added to the medium such that the
concentration of luciferase mRNA in the medium was 0.3 or 1
.mu.g/ml, and the mixture was stood in a 5% CO.sub.2 incubator at
37.degree. C. for 72 hr. As a control sample of T cell activation
stimulation, T cells supplemented with TransAct Miltenyi Biotec
company) and Dynabeads (Thermo Fisher Scientific company) were also
prepared. Luciferase expressed in T cells was measured using
Bright-Glo Luciferase Assay System Kit (Promega). The number of
viable T cells was measured using CellTiter-Glo kit (Promega). The
expression level of Luciferase is shown in FIG. 3(I), and the
survival number of T cells is shown in FIG. 3(II) and (III).
11. Preparation of Luc mRNA-Encapsulating Lipid Nanoparticles
[0314] A lipid mixture containing compound 35 as cationic lipid
(compound 35:DPPC:Cholesterol:SUNBRIGHT
GM-020:SUNBRIGHT=60:10.6:28:1.4 molar ratio) was dissolved in 90%
EtOH, 10% water to give a 8.1 mg/ml lipid solution. On the other
hand, luciferase mRNA (Luc mRNA) (TriLink company) was dissolved in
2-morpholinoethanesulfonic acid (MES) buffer (pH 5.0) to give 0.18
mg/ml mRNA solution. The obtained lipid solution and mRNA solution
were mixed at room temperature by a Nanoassemblr apparatus
(Precision Nanosystems) at a flow rate ratio of 3 ml/min:6 ml/min
to give a dispersion of Luc mRNA-encapsulating lipid nanoparticles.
The obtained dispersion was dialyzed using Slyde-A-Lyzer (molecular
weight 20k for fraction, Thermo Scientific) against water at room
temperature for 1 hr, and against PBS at 4.degree. C. for 48 hr.
Successively, the dialysate was filtered through a 0.2 .mu.m
syringe filter (Iwaki) and stored at 4.degree. C.
12. Particle Size Measurement of Luc mRNA-Encapsulating Lipid
Nanoparticles
[0315] To 1 .mu.l of a Luc mRNA-encapsulating lipid nanoparticles
stock solution was added 99 .mu.l of phosphate buffered saline (137
mM NaCl, 7.99 mM Na.sub.2HPO.sub.4, 2.7 mM KCl, 1.47 mM
KH.sub.2PO.sub.4, pH 7.4). The obtained dispersion was subjected to
dynamic light scattering measurement using Zetasizer Nano ZS
(Malvern instruments), and the cumulant analysis of the
autocorrelation function was performed to measure the Z average
particle size and the polydispersity index.
13. .lamda. Potential Measurement of Luc mRNA-Encapsulating Lipid
Nanoparticles
[0316] To 50 .mu.l of a Luc mRNA-encapsulating nanoparticle stock
solution was added 700 .mu.l of HEPES buffer (10 mM HEPES-Na0H, pH
7.3). The obtained dispersion was subjected to electrophoretic
light scattering measurement using Zetasizer Nano ZS (Malvern
instruments) to measure the potential.
14. Measurement of Encapsulation Rate and Concentration of Mrna for
Luc Mrna-Encapsulating Lipid Nanoparticles
[0317] A Luc mRNA-encapsulating nanoparticle stock solution was
diluted with TE buffer to adjust the mRNA concentration to about 4
.mu.g/ml. As an mRNA concentration standard solution, naked mRNA
was diluted with TE buffer to 4 .mu.g/ml. The diluted Luc
mRNA-encapsulating nanoparticles and naked mRNA concentration
standard solution (each 60 p1) were each mixed with 60 .mu.l of TE
buffer or TE buffer containing 2% Triton-X100. The mixture was
allowed to stand at room temperature for 5 min, mixed with 120
.mu.l of Quant-iT.TM. RiboGreen (registered trade mark), and the
mixture was further allowed to stand for 5 min. The fluorescence
intensity of the mixture was measured using Envision microplate
reader (Perkin-Elmer company). The mRNA encapsulation rate and mRNA
concentration were calculated by the following formulas.
% mRNA encapsulation rate=(1-F.sub.TE/E.sub.Triton).times.100
mRNA concentration=(F.sub.Triton-b).times.d/m
(wherein F.sub.TE shows RiboGreen fluorescence intensity of lipid
nanoparticles mixed with TE buffer, F.sub.rriton shows RiboGreen
fluorescence intensity of lipid nanoparticles mixed with TE buffer
containing 2% Triton-X100, b and m show y-intercept and slope
obtained from the calibration curve of the concentration standard
siRNA, and d is the dilution rate of lipid nanoparticles)
[0318] The analysis results of the Luc mRNA-encapsulating lipid
nanoparticles are shown in Table 2.
TABLE-US-00002 TABLE 2 Z- average mRNA particle polydispersity zeta
encapsulation composition size index potential rate compound 88 nm
0.029 -0.2 mV 96% 35-luc mRNA
15. Highly Efficient mRNA Transfection into Human Peripheral Blood
CD3 Positive Pan T Cells by Co-Addition of Activation Stimulant and
Lipid Nanoparticles
[0319] Human peripheral blood CD3 positive pan T cells (Precision
Bioservices) were seeded on a round-bottomed 96-well plate
(Corning) at a cell density of 1.times.10.sup.5 cells/well. A
serum-free hematopoietic cell medium X-VIVO10 (Lonza) supplemented
with 30 ng/ml recombinant IL-2 (Thermo Fisher Scientific), and with
TransAct (Milteny Biotech) or Dynabeads Human T-Activator CD3/CD28
(ThermoFisher Scientific), which stimulates activation of T cells,
according to the protocol recommended by each manufacturer, was
used as the medium. Subsequently, lipid nanoparticle compound
35-luc mRNA encapsulating luciferase mRNA (TriLink) were added to
the medium such that the concentration of luciferase mRNA in the
medium was 0.1, 0.3 or 1 .mu.g/ml, and the mixture was stood in a
5% CO.sub.2 incubator at 37.degree. C. for 72 hr. Luciferase
expressed in T cells was measured using Bright-Glo Luciferase Assay
System Kit (Promega). The survival and proliferation rate of T
cells was measured using CellTiter-Glo Luminescent Cell Viability
Assay kit (Promega KK). The obtained results are shown in FIGS. 4
and 5. It was shown that addition of lipid nanoparticles
encapsulating Luc mRNA to T cells under activation stimulation
dramatically improves transfection activity (FIG. 4). In addition,
the survival and proliferation rate of T cells was maintained at a
high level (FIG. 5).
16. Highly Efficient Luc mRNA Transfection into Human CD4/CD8
Positive T Cells by Co-Addition of Activation Stimulant and Lipid
Nanoparticles
[0320] Human peripheral blood leukocyte fraction Leukopak
(HemaCare) was washed with LOVO Cell Processing System (Fresenius),
and CD4 and CD8 positive cells were collected with cell processing
system CliniMACS (Milteny Biotec). The obtained CD4/CD8 positive
cells were seeded on a flat-bottomed 96-well plate (Corning) at a
cell density of 1.times.10.sup.5 cells/well. For cell culture, a
medium supplemented with 26 mL of OPTmizer CTS T-cell Expansion
Supplement, 20 mL of CTS Immune SR, 10 mL of L-Glutamine 200 mM
(all ThermoFischer Scientific), 10 mL of Streptomycin Sulfate 10
mg/ml (MEIJI company), and 4.2 ng/ml MACS GMP Recombinant Human
IL-2 (Milteny Biotec), per 1 L of OPTmizer CTS T-Cell Expansion
Basal medium, was used. In addition, as a T cell activation
stimulant, TransAct (Milteny Biotech) was added according to the
protocol recommended by the manufacturer. Subsequently, lipid
nanoparticle compound 35-luc mRNA encapsulating luciferase mRNA
(TriLink) were added to the medium such that the concentration of
luciferase mRNA in the medium was 1, 3 or 10 ug/ml, and the mixture
was stood in a 5% CO.sub.2 incubator at 37.degree. C. for 72 hr.
Luciferase expressed in T cells was measured using Bright-Glo
Luciferase Assay System Kit (Promega). The survival and
proliferation rate of T cells was measured using CellTiter-Glo
Luminescent Cell Viability Assay kit (Promega KK). The obtained
results are shown in FIG. 6. It was shown that addition of lipid
nanoparticles encapsulating Luc mRNA to T cells under activation
stimulation dramatically improves transfection activity. In
addition, the survival and proliferation rate of T cells was
maintained at a high level.
INDUSTRIAL APPLICABILITY
[0321] Using the nucleic acid delivery carrier of the present
invention or the nucleic acid delivery method of the present
invention, a step of activating/proliferating T cells and a step of
introducing a gene into T cells can be performed simultaneously in
one pod. As a result, an agent for immune cell therapy can be
provided in a short period of time at a low production cost, and
the present invention is extremely useful since an immunocyte
therapy can be provided at a lower cost.
[0322] This application is based on a patent application No.
2018-197069 filed in Oct. 18, 2018 and a patent application No.
2019-124629 filed in Jul. 3, 2019, the contents of which are hereby
incorporated by reference in full herein.
* * * * *
References